Modulation of gene expression by combination therapy

ABSTRACT

The invention relates to the modulation of gene expression. In particular, the invention relates to compositions comprising antisense oligonucleotides which inhibit expression of a gene in operable association with protein effectors of a product of that gene, and methods of using the same.  
     In addition, the invention relates to the modulation of mammalian gene expression regulated by methylation.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to the modulation of gene expression, andto the modulation of mammalian gene expression regulated by methylation.

[0003] 2. Summary of the Related Art

[0004] The modulation of expression of genes has long been pursued byresearchers. For example, many mammalian diseases are associated withthe over- or under-expression of certain genes. By modulating theexpression of the gene whose aberrant expression is associated with adisease (or a predisposition to develop such a disease), the diseasesymptoms may be alleviated.

[0005] For example, it would be desirable to modulate the enzyme, DNAMethyltransferase (DNA MeTase), which mediates the methylation ofmammalian DNA.

[0006] Methylation of mammalian DNA is enzymatically mediated by thecovalent modification of the fifth carbon position of the pyrimidinering of cytosine in CpG dinudeotides. Changes in the pattern of DNAmethylation have been correlated with a number of processes ineukaryotes. Holliday (1990) Philos. Trans. R. Soc. Lond. B. Biol. Sci.326: 329-338 discusses the role of methylation in parental imprinting.Antequera et al. (1989) Cell 58: 509-517 discusses the significance ofmethylation in developmental regulation. Fedoroff et al. (1989) Cell 56:181-191 discloses that methylation is involved in transposition. Hareand Taylor (1985) Proc. Natl. Acad. Sci. USA 82: 7350-7354 disclosesthat methylation is also implicated in DNA repair. In addition, Gartlerand Riggs (1983) Ann. Rev. Genet. 17: 155-190 correlates methylationwith X chromosome inactivation, while Bird et al. (1986) Nature 321:209-213 discloses that methylation also plays a pivotal role inchromatin organization.

[0007] While several observations have suggested a role for DNAmethylation in cancer pathogenesis, there has been a great deal ofdisagreement as to the mechanisms involved. Szyf et al. (1996)Pharmacol. Ther. 70(1): 1-37 discloses that methylation anddemethylation activities are critical components of some tumorigenicgrowth forms. MacLeod and Szyf (1995) J. Biol. Chem. 270: 8037-8043discloses that the level of DNA methyltransferase activity may be anodal control point over oncogenic growth. On the other hand, othershave dismissed the theory that DNA Methyltransferase plays a causal rolein oncogenesis.

[0008] A significant obstacle to the investigation of the role ofmethylation lies in the deficiencies of available methods to modulatemethylation itself. To date, most studies have relied on 5-aza-C and/or5-aza-dC to inhibit DNA methylation by forming a stable adduct with DNAmethyltransferase, thus mimicking the transient covalent intermediatecomplex believed to be formed during methylation (see, e.g., Wu andSanti (1987) J. Biol. Chem. 262: 4778-4786). This approach, albeiteffective in reducing DNA methyltransferase activity and correlatingwith tumorigenesis inhibition, is therapeutically deficient.Unfortunately, threshold concentrations of 5-azaC or 5-aza-dCempirically necessary to inhibit DNA methyltransferase activity havebeen found to be toxic to mammals (Juttermann et al. (1994) Proc. Natl.Acad. Sci. USA 91: 11797-11801; Laird (1997) Mol. Med. Today 3:223-229).

[0009] More recently, Szyf et al. (1995) J. Biol. Chem. 267:12831-12836, has disclosed a more promising approach to the modulationof DNA methylation using expression of antisense RNA complementary tothe DNA methyltransferase mRNA to study the effect of methylation oncancer cells. U.S. Pat. No. 5,578,716, discloses the use of antisenseoligonudeotides complementary to the DNA methyltransferase gene toinhibit gene expression. These developments have provided powerful newtools for probing the role of methylation in numerous cellularprocesses. In addition, they have provided promising new approaches fordeveloping therapeutic compounds that can modulate methylation levels.

[0010] The effect of antisense inhibition is not immediate, due to thehalf-life of the DNA methyltransferase enzyme. Thus, although theexpression of DNA methyltransferase is modulated, residual enzyme cancontinue to methylate DNA until such enzyme is degraded. Furthermore,polysome-associated DNA methyltransferase mRNA may also persist for sometime, allowing additional translation to produce additional enzyme. Inaddition, the pharmacodynamic properties of oligonucleotides suggestthat lower doses than are currently used could be beneficial (Agrawal etal. (1995) Clinical Pharmacokinetics 28: 7-16; Zhang et al. (1995)Clinical Pharmacology and Therapeutics 58: 44-53).

[0011] Therefore, there remains a need to develop more effective methodsfor the modulation of the expression of genes, such as DNAmethyltransferase, that would reduce the required dosage of antisenseoligonucleotides, specific inhibitors of gene products, or other agentscurrently used while at the same time effectively accomplish theinhibition of gene expression.

SUMMARY OF THE INVENTION

[0012] The present inventors have devised a combination approach bywhich a gene, such as mammalian DNA methyltransferase (DNA MeTase), isinhibited at the genetic level as well as at the protein level.Surprisingly, this combination approach has been found to reduce therequired dosage for efficacy of both antisense oligonucleotides againstthe gene as well as small molecule inhibitors of the gene product.

[0013] The invention also provides improved methods and compositions forthe modulation of mammalian DNA MeTase at the genetic level as well asat the protein level and the modulation of mammalian gene expressionregulated by methylation. The methods and compositions according to theinvention are useful as analytical tools for transgenic studies and astherapeutic tools.

[0014] In a first aspect the invention provides a method for inhibitingthe expression of a gene in a cell comprising contacting the cell withan effective synergistic amount of an antisense oligonucleotide whichinhibits expression of the gene, and an effective synergistic amount ofa protein effector of a product of the gene.

[0015] In embodiments of the first aspect of the invention, the cell iscontacted with an effective synergistic amount of at least one antisenseoligonucleotide for an effective period of time. In certain embodiments,the cell is contacted with an effective synergistic amount of at leastone protein effector for an effective period of time. In certainembodiments, each of the antisense oligonudeotide and the proteineffector is admixed with a pharmaceutically acceptable carrier mediumprior to contacting the cell. In certain embodiments, the antisenseoligonucleotide and the protein effector are mixed prior to contactingthe cell.

[0016] In certain preferred embodiments of the first aspect of theinvention, the cell is contacted separately with each of the antisenseoligonucleotide and the protein effector. In certain embodiments, thecell is contacted with the antisense oligonucleotide prior to beingcontacted with the protein effector. In certain embodiments, the geneencodes a DNA methyltransferase and the cell contacted with theantisense oligonucleotide prior to being contacted with the proteineffector is induced to undergo apoptosis or is arrested in the S phaseof the cell cycle. In certain embodiments, the cell is contacted withthe protein effector prior to being contacted with the antisenseoligonucleotide. In certain embodiments, the gene encodes a DNAmethyltransferase and the cell contacted with the protein effector priorto being contacted with the antisense oligonucleotide is arrested in theG₁ phase of the cell cycle.

[0017] In certain preferred embodiments of the first aspect of theinvention, the gene encodes a DNA methyltransferase and the cellcomprises a gene whose expression has been inactivated by methylation.In certain embodiments, expression of the gene whose expression has beeninactivated by methylation is reactivated in the contacted cell. Inpreferred embodiments, the gene whose expression has been inactivated bymethylation is the p16^(ink4) tumor suppressor gene.

[0018] In a second aspect, the invention provides a method for treatinga disease responsive to inhibition of a gene. The method according tothis aspect of the invention includes administering to a mammal,including a human, which has at least one cell affected by the disease,a therapeutically effective synergistic amount of an antisenseoligonucleotide which inhibits expression of the gene, and atherapeutically effective synergistic amount of a protein effector of aproduct of the gene.

[0019] In embodiments of the second aspect of the invention, the mammalis administered a therapeutically effective synergistic amount of atleast one antisense oligonudeotide for a therapeutically effectiveperiod of time. In certain embodiments, the mammal is administered atherapeutically effective synergistic amount of at least one proteineffector for a therapeutically effective period of time. In certainembodiments, each of the antisense oligonucleotide and the proteineffector is admixed with a pharmaceutically acceptable carrier mediumprior to administration to the mammal. In certain embodiments, theantisense oligonucleotide and the protein effector are mixed prior toadministration to the mammal.

[0020] In certain preferred embodiments of the second aspect of theinvention, the the antisense oligonucleotide and the protein effectorare separately administered to the mammal. In certain embodiments, theantisense oligonucleotide is administered to the mammal prior to theadministration of the protein effector. In certain embodiments, the geneencodes a DNA methyltransferase and the cell affected by the disease inthe mammal to which the the antisense oligonucleotide is administeredprior to the administration of the protein effector is induced toundergo apoptosis or is arrested in the S phase of the cell cycle. Incertain embodiments, the protein effector is administered to the mammalprior to the administration of the antisense oligonucleotide. In certainembodiments, the gene encodes a DNA methyltransferase and the cellaffected by the disease in the mammal to which the protein effector isadministered prior to the administration of the antisenseoligonucleotide is arrested in the G₁ phase of the cell cycle.

[0021] In certain preferred embodiments of the second aspect of theinvention, the gene encodes a DNA methyltransferase and the cellaffected by the disease comprises a gene whose expression has beeninactivated by methylation. In certain embodiments, expression of thegene whose expression has been inactivated by methylation is reactivatedin the cell in the mammal to which has been administered atherapeutically effective synergistic amount of an antisenseoligonucleotide and a therapeutically effective synergistic amount of aprotein effector. In preferred embodiments, the gene whose expressionhas been inactivated by methylation is the p16^(ink4) tumor suppressorgene.

[0022] In a third aspect, the invention provides a method for inhibitingtumor growth in a mammal. The method according to this aspect of theinvention includes administering to a mammal, including a human, whichhas at least one neoplastic cell present in its body a therapeuticallyeffective synergistic amount of an antisense oligonudeotide whichinhibits expression of a gene involved in tumorigenesis, and atherapeutically effective synergistic amount of a protein effector of aproduct of the gene.

[0023] In embodiments of the third aspect of the invention, the mammalis administered a therapeutically effective synergistic amount of morethan one antisense oligonucleotide for a therapeutically effectiveperiod of time. In certain embodiments, the mammal is administered atherapeutically effective synergistic amount of at least one proteineffector for a therapeutically effective period of time. In certainembodiments, each of the antisense oligonucleotide and the proteineffector is admixed with a pharmaceutically acceptable carrier mediumprior to administration to the mammal. In certain embodiments, theantisense oligonucleotide and the protein effector are mixed prior toadministration to the mammal.

[0024] In certain preferred embodiments of the third aspect of theinvention, the the antisense oligonucleotide and the protein effectorare separately administered to the mammal. In certain embodiments, theantisense oligonucleotide is administered to the mammal prior to theadministration of the protein effector. In certain embodiments, the geneencodes a DNA methyltransferase and the neoplastic cell in the mammal towhich the the antisense oligonucleotide is administered prior to theadministration of the protein effector is induced to undergo apoptosisor is arrested in the S phase of the cell cycle. In certain embodiments,the protein effector is administered to the mammal prior to theadministration of the antisense oligonucleotide. In certain embodiments,the gene encodes a DNA methyltransferase and the neoplastic cell in themammal to which the protein effector is administered prior to theadministration of the antisense oligonucleotide is arrested in the G₁phase of the cell cycle.

[0025] In certain preferred embodiments of the third aspect of theinvention, the gene encodes a DNA methyltransferase and the neoplasticcell comprises a gene whose expression has been inactivated bymethylation. In certain embodiments, expression of the gene whoseexpression has been inactivated by methylation is reactivated in theneoplastic cell in the mammal to which has been administered atherapeutically effective synergistic amount of an antisenseoligonudeotide and a therapeutically effective synergistic amount of aprotein effector. In preferred embodiments, the gene whose expressionhas been inactivated by methylation is the p16^(ink4) tumor suppressorgene.

[0026] In certain embodiments of the first three aspects of theinvention, the gene encodes a DNA methyltransferase. In certainembodiments, the protein effector is selected from the group consistingof 5-aza-cytidine, 5-aza-2′-deoxycytidine, 5-fluoro-2′-deoxycytidine and5,6-dihydro-5-azacytidine. In certain embodiments of the first, second,and third aspects, the gene encodes a histone deacetylase. In certainembodiments, the protein effector is selected form the group consistingof trichostatin A, depudecin, trapoxin, suberoylanilide hydroxamic acid,FR901228, MS-27-275, CI-994, and sodium butyrate. In certain embodimentsof the first three aspects, the gene encodes a thymidylate synthase. Incertain embodiments, the protein effector is selected form the groupconsisting of 5-fluorouracil, Tomudex, Raltitrexed, Zeneca ZD1694,Zeneca ZD9331, Thymitaq, AG331, Ly231514, and BW1843U89.

[0027] In various embodiments of the first three aspects of theinvention, the antisense oligonucleotide is in operable association witha protein effector. In certain embodiments, the antisenseoligonucleotide has at least one internucleotide linkage selected fromthe group consisting of phosphorothioate, phosphorodithioate,alkylphosphonate, alkylphosphonothioate, phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphorothioate and sulfone internucleotidelinkages. In certain embodiments, the antisense oligonucleotide is achimeric oligonucleotide comprising a phosphorothioate, phosphodiesteror phosphorodithioate region and an alkylphosphonate oralkylphosphonothioate region. In certain embodiments, the antisenseoligonucleotide comprises a ribonucleotide or 2′-O-substitutedribonucleotide region and a deoxyribonucleotide region.

[0028] In a fourth aspect, the invention provides an inhibitor of a genecomprising an antisense oligonucleotide which inhibits expression of thegene in operable association with a protein effector of a product of thegene. In certain embodiments of this aspect of the invention, theantisense oligonucleotide is in operable association with two or moreprotein effectors.

[0029] In certain embodiments of the fourth aspect of the invention, thegene encodes a DNA methyltransferase. In certain embodiments, theprotein effector is selected from the group consisting of5-aza-cytidine, 5-aza-2′-deoxycytidine, 5-fluoro-2′-deoxycytidine and5,6-dihydro-5-azacytidine. In certain embodiments, the gene encodes ahistone deacetylase. In certain embodiments, the protein effector isselected form the group consisting of trichostatin A, depudecin,trapoxin, suberoylanilide hydroxamic acid, FR901228, MS-27-275, CI-994,and sodium butyrate. In certain embodiments, the gene encodes athymidylate synthase. In certain embodiments, the protein effector isselected form the group consisting of 5-fluorouracil, Tomudex,Raltitrexed, Zeneca ZD1694, Zeneca ZD9331, Thymitaq, AG331, Ly231514,and BW1843U89.

[0030] In certain embodiments of the fourth aspect of the invention, theantisense oligonucleotide has at least one internucleotide linkageselected from the group consisting of phosphorothioate,phosphorodithioate, alkylphosphonate, alkylphosphonothioate,phosphotriester, phosphoramidate, siloxane, carbonate,carboxymethylester, acetamidate, carbamate, thioether, bridgedphosphoramidate, bridged methylene phosphonate, bridged phosphorothioateand sulfone internucleotide linkages. In certain embodiments, theantisense oligonucleotide is a chimeric oligonucleotide comprising aphosphorothioate, phosphodiester or phosphorodithioate region and analkylphosphonate or alkylphosphonothioate region. In certainembodiments, the antisense oligonucleotide comprises a ribonucleotide or2′-O-substituted ribonucleotide region and a deoxyribonucleotide region.

[0031] In a fifth aspect, the invention provides a pharmaceuticalcomposition comprising an inhibitor of a gene comprising an antisenseoligonucleotide which inhibits expression the gene in operableassociation with a protein effector of a product of the gene. In certainembodiments, the composition further comprises a pharmaceuticallyacceptable carrier.

[0032] In a sixth aspect, the invention provides a method forinvestigating the role of a gene and/or a product of the gene incellular growth, including the growth of tumor cells. In the methodaccording to this aspect of the invention, the cell type of interest iscontacted with a synergistic amount of an antisense oligonudeotide whichinhibits expression the gene and a synergistic amount of a proteineffector of a product of the gene, as described for the first aspectaccording to the invention, resulting in inhibition of expression of thegene in the cell. In certain embodiments, the gene encodes the productselected from the group consisting of a DNA methyltransferase, a histonedeacetylase, and a thymidylate synthase. The combinations describedherein may be administered at different points in the cell cycle, or inconjunction with promoters or inhibitors of cell growth to determine therole of the gene and/or the product of the gene in the growth of thecell type of interest.

[0033] The methods, inhibitors, and compositions of the invention thatinhibit expression and/or activity of a gene and/or gene product may beused to treat patients having, or predisposed to developing, a diseaseresponsive to inhibition of the gene. For example, an inhibitor orcomposition of the invention may administered with apharmaceutically-acceptable carrier (e.g., physiological sterile salinesolution) via any route of administration to patient suffering from adisease responsive to inhibition of a gene in an attempt to alleviateany resulting disease symptoms. For example, an inhibitor or compositionof the invention may be used to relieve symptoms of cancer in a patientsuffering from cancer, one exemplary, non-limiting disease responsive toinhibition of DNA methyltransferase. Pharmaceutically-acceptablecarriers and their formulations are well-known and generally describedin, for example, Remington's Pharmaceutical Sciences (18th Edition, ed.A. Gennaro, Mack Publishing Co., Easton, Pa., 1990).

BRIEF DESCRIPTION OF THE DRAWINGS

[0034]FIG. 1 is a diagrammatic representation of the DNAmethyltransferase gene (above) and the positions of the various 20-merphosphorothioate antisense oligonucleotides (shown as filled circles) onthe gene, with the positions of two non-limiting syntheticoligonucleotides, MG88 and MG98, highlighted.

[0035]FIG. 2 is a representation of autoradiographs of Western blottinganalyses showing that treatment with 40 nM or 80 nM of a representative,non-limiting antisense oligonucleotide, MG88, results in an inhibitionof MeTase protein expression in both A549 and T24 cells.

[0036]FIG. 3A is a representation of autoradiographs of a series ofimmunoprecipitations followed by Western blotting analyses of T24 celllysates showing that p16^(ink4) protein levels (upper panel) increase asDNA MeTase protein levels (lower panel) decrease following treatment ofthe cells for 3, 5, 8, or 10 days with 40 or 75 nM of a-representative,non-limiting antisense oligonucleotide of the invention, MG88, whereHeLa cells served as a positive control for p16^(ink4) proteinexpression.

[0037]FIG. 3B is a graphic representation of p16^(ink4) protein levelsnormalized to cell number of T24 cells treated with 40 nM or 75 nM MG88for 3, 5, 8, and 10 days.

[0038]FIG. 4 is a representation of an autoradiograph of a Westernblotting analysis of T24 cells treated with 40 nM or 75 nM of arepresentative non-limiting antisense oligonucleotide of the invention,MG88, using an antibody recognizing all phosphorylated forms of Rb.

[0039]FIG. 5 is a representation of autoradiographs of a series ofWestern blotting analyses of T24 cell lysates prepared 3, 5, or 7 daysafter cessation of a 10 day treatment of the cells with lipofectin only,40 nM of MG88, or 40 nM or 75 nM of control oligonucleotide MG208,demonstrating that between 5-7 days post-treatment, DNA MeTase proteinexpression is restored and p16^(ink4) protein expression is diminished.

[0040]FIG. 6 is a graphic representation of the quantitation of DNAMeTase and p16^(ink4) protein levels in T24 cells during ten days oftreatment with MG88 and 7 days (i.e., days 11-17) post-treatmentperiods, demonstrating the inverse relationship between p16 proteinlevels and DNA MeTase protein levels in these cells.

[0041]FIG. 7 is a representation of a series photographs of showing themethylation-specific PCR (MSP) products resolved on 2% agarose gels ofthe p16^(ink4) gene promoter from T24 cells treated with 40 or 75 nM ofMG88 or MG208 of 3, 5, 8, or 10 days using PCR primers specific formethylated p16^(ink4) (M lanes) or unmethylated p16^(ink4) (U lanes),demonstrating that demethylation of p16^(ink4) occurred only in MG88treated cells after at least 3 days of treatment.

[0042]FIG. 8 is a diagrammatic representation of the p16^(ink4) proximalpromoter from T24 cells showing the methylation patterns after 0, 3, or5 days of treatment of the cells with MG88 (left) or controloligonucleotide MG208 (right), demonstrating that reduced methylationoccurred only with MG88 treatment. Day 3 post-treatment methylationpatterns are shown at the bottom of the figure.

[0043]FIG. 9A is a representation of a photograph of the results ofbisulfite sequencing of the p16^(ink4) gene promoter inp16^(ink4)-expressing clone MG88 C4-5 30 days following cessation of a 5day treatment with 75 nM MG88, demonstrating that all CpG sitesevaluated were not methylated in this clone even after 30 days inculture post-MG88 treatment.

[0044]FIG. 9B is a graphic representation showing a growth curve of T24cells during treatment (days 0-5) and post-treatment (days 6-18) withlipofectin only (squares), 75 nM MG88, a non-limiting representativeantisense oligonudeotide of the invention (circles), or 75 nM or controloligonucleotide, MG208 (triangles), demonstrating the anti-proliferativeeffect of MG88.

[0045]FIG. 9C is a graphic representation showing a growth curve duringpost-treatment of T24 cell clones following a five day treatment withMG88 (clone 4-5, triangles), MG208 (clone 2-4, squares), or lipofectinonly (clone 5, diamonds). The inserted representation of twoautoradiographs show the p16^(ink4) protein level in each of the clonesof days 36 and 49 post-treatment.

[0046]FIG. 10A is a representation of autoradiographs of Westernblotting analyses of p21^(WAF1), MeTase, and α-actin protein levels inT24 cells treated for 24 hours (left panels) or 48 hours (right panels)with lipofectin only, or 40 nM or 75 nM of MG88 or MG208, demonstratingthat p²¹ ^(WAF1) protein expression is induced by inhibition of MeTaseexpression.

[0047]FIG. 10B is a representation of autoradiographs of Westernblotting analyses showing the dose-response of p21^(WAF1) protein levelsin T24 cells treated for 24 hours with 20 nM, 40 nM, or 80 nM of MG88 orMG208. Actin protein levels are shown as a control for protein loading.

[0048]FIG. 11 is a representative of an autoradiograph showing aNorthern blotting analysis of RNA from T24 cells treated for 24 or 48hours with MG88 or MG208.

[0049]FIG. 12 is a representation of an autoradiograph of a Westernblotting analysis showing the reactivation of p16 expression in T24cells following treatment for three days with increasing concentrationsof a representative, non-limiting DNA MeTase protein effector, 5-aza-dC.

[0050]FIG. 13 is a representation of an autoradiograph of the Westernblot analysis showing the synergistic reactivation of p16 expression inT24 cells following treatment for three days with a representative,nonlimiting, synthetic antisense oligonucleotide (MG88) and/or arepresentative non-limiting DNA MeTase protein effector (5-aza-dC)according to the invention; the three panels show varying combinationsand concentrations of representative antisense oligonucleotides and DNAMeTase protein effectors according to the invention.

[0051]FIG. 14 is a representation of an autoradiograph of the Westernblot analysis showing the synergistic reactivation of p16 expression inT24 cells following treatment for three days with a representative,nonlimiting, synthetic antisense oligonucleotide (MG98) and/or arepresentative non-limiting DNA MeTase protein effector (5-aza-dC)according to the invention; the three panels show varying combinationsand concentrations of representative oligonucleotides and DNA MeTaseprotein effectors according to the invention.

[0052]FIG. 15 is a representation of an autoradiograph of the Westernblot analysis showing the synergistic reactivation of p16 expression inT24 cells following treatment for three days with a representative,nonlimiting, synthetic oligonucleotide (MG88) and/or a representativenon-limiting DNA MeTase protein effector (5-aza-dC) according to theinvention at low concentrations; the three panels show varyingcombinations and concentrations of representative oligonucleotides andDNA MeTase protein effectors according to the invention.

[0053]FIG. 16 is a graphic representation showing the ability of arepresentative, nonlimiting, synthetic antisense oligonucleotide (MG98)and of a representative, nonlimiting, DNA MeTase protein effector(5-aza-dC) according to the invention to inhibit T24 human bladdercancer cell growth in a synergistic fashion resulting in an increasedinhibitory effect as compared to that observed using either only theantisense oligonucleotides or only the DNA MeTase protein effectors.

[0054]FIG. 17 is a graphic representation showing the synergisticinhibition of T24 human bladder cancer cell growth after treatment forseven days with lipofectin only (first bar from the left); 1 μM of arepresentative, nonlimiting, DNA MeTase protein effector, 5-aza-dC(second bar from the left); 40 nM of control synthetic oligonucleotideMG207 (third bar from the left); 40 nM of a representative nonlimitingsynthetic MeTase antisense oligonucleotide, MG98 (fourth bar from theleft); MG207 plus 5-aza-dC (fifth bar from the left); or MG98 plus5-aza-dC (sixth bar from the left).

[0055]FIG. 18 is a graphic representation showing the ability of arepresentative, nonlimiting, synthetic oligonucleotide (MG98) and of arepresentative, nonlimiting, DNA MeTase protein effector (5-aza-dC)according to the invention to inhibit A549 human non-small cell lungcancer cell growth in a synergistic fashion resulting in an increasedinhibitory effect as compared to that observed using either only theoligonudeotides or only the DNA MeTase protein effectors.

[0056]FIG. 19 is a graphic representation showing the ability of arepresentative, nonlimiting, synthetic oligonucleotide (MG98) and of arepresentative, nonlimiting, DNA MeTase protein effector (5-aza-dC)according to the invention to inhibit Colo 205 human colon cancer cellgrowth (expressed as tumor volume over time) in a synergistic fashionresulting in an increased inhibitory effect as compared to that observedusing either only the oligonucleotides or only the DNA MeTase proteineffectors.

[0057]FIG. 20A is a graphic representation showing the inhibition ofColo 205 tumor cell growth in nude mice following treatment of the micewith saline (diamond); 0.5 mg/kg of a representative, nonlimiting MeTasesynthetic oligonucleotide, MG98 (square); 0.1 mg/kg of a representative,nonlimiting, DNA MeTase protein effector, 5-aza-dC (triangle); or acombination of both MG98 plus 5-aza-dC (X).

[0058]FIG. 20B is a graphic representation showing the inhibition ofColo 205 human colon cancer cell growth (expressed as final tumorvolume) by a representative, nonlimiting, synthetic oligonucleotide(MG98) and by a representative, nonlimiting DNA MeTase protein effector(5-aza-dC) according to the invention, in a synergistic fashion,resulting in an statistically increased inhibitory effect (p<0.05) ascompared to that observed using either only oligonucleotide, proteineffector, or saline.

[0059]FIG. 21 is a schematic diagram showing a series of FACS histogramanalyses of T24 cells treated with a representative, nonlimiting,synthetic oligonucleotide (MG88) and/or a representative non-limitingDNA MeTase protein effector (5-aza-dC) according to the invention, atdifferent schedules. The upper panel of histograms shows cells treatedon schedule A, where MG88 is administered before 5-aza-dC. The lowerpanel of histograms shows cells treated on schedule B, where 5-aza-dC isadministered before MG88.

[0060]FIG. 22 is a representation of an autoradiograph of the Westernblotting analysis showing the synergistic inhibition of thymidylatesynthase protein expression in T24 cells using the combination of arepresentative, nonlimiting, synthetic antisense oligonudeotide (MG2605)and a representative, nonlimiting TS protein effector (5-FU) accordingto the invention.

[0061]FIG. 23 is a schematic diagram showing a series of FACS histogramanalyses of T24 cells treated with a representative, nonlimitingsynthetic thymidylate synthase antisense oligonucleotide and/or arepresentative nonlimiting TS protein effector (5-FU) according to theinvention. The top histograms shows cells treated with lipofectin only;the second histogram shows cells treated with 25 nM mismatch controloligonucleotide; the third histogram from the top shows cells treatedwith 25 nM of the TS antisense oligonucleotide, MG2605; the fourthhistogram from the top shows cells treated with 500 nM of 5-FU; thefifth histogram from the top shows cells treated with 5-FU plus mismatcholigonucleotide; and the sixth histogram (i.e., the bottom histogram)shows cells treated with 5-FU plus the TS antisense oligonucleotide,MG2605.

[0062]FIG. 24A is a graphic representation showing the percentages ofT24 cells in the G₁ phase (gray bars; section M2 on inserted histograms)S phase (black bars; section M3 on inserted histograms), and G₂/M phase(white bars; section M4 on inserted histogram) following treatment with25 nM of a representative, nonlimiting TS antisense oligonucleotideMG2605, 25 nM of control oligonucleotide MG2606, 5 μM of arepresentative nonlimiting TS protein effector, 5-FU, or a combinationof oligonucleotide plus 5-FU.

[0063]FIG. 24B is a graphic representation of the number of T24 cellsremaining following treatment with following no treatment; or treatmentwith 25 nM of a representative, nonlimiting TS antisense oligonucleotideMG2605; 25 nM of control oligonucleotide MG2606; 5 μM of arepresentative nonlimiting TS protein effector, 5-FU; or a combinationof MG2605 or MG2606 plus 5-FU.

[0064]FIG. 25 is a representation of an autoradiograph of the Westernblotting analysis showing the synergistic induction of p21^(WAF1) by thecombination of a representative, nonlimiting, synthetic HDAC antisenseoligonucleotide and a representative, nonlimiting HDAC protein effector(TSA) according to the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] The present inventors have devised a combination approach bywhich expression of a gene is inhibited at the genetic level as well asat the protein level. Surprisingly, this combination approach has beenfound to reduce the required dosage for efficacy of both antisenseoligonucleotides against the gene itself as well as of protein effectorsagainst the product of the gene.

[0066] The invention provides improved methods, compounds (e.g.,inhibitors such as antisense oligonucleotides and protein effectors) andcompositions for the modulation of gene expression at the genetic levelas well as at the protein level. In addition, where the gene is DNAmethyltransferase (DNA MeTase), the invention provides methods,compounds, and compositions for the modulation of mammalian geneexpression regulated by methylation. The methods and compositionsaccording to the invention are useful as analytical tools for transgenicstudies and as therapeutic tools, including as gene therapy tools. Theinvention also provides methods and compositions which may bemanipulated and fine tuned to fit the condition(s) to be treated whileproducing fewer side effects. Standard reference works setting forth thegeneral principles of recombinant DNA technology include Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, New York(1994); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1989); Kauftnan etal., Eds., Handbook of Molecular and Cellular Methods in Biology andMedicine, CRC Press, Boca Raton (1995); McPherson, Ed., DirectedMutagenesis: A Practical Approach, IRL Press, Oxford (1991). The patentsand scientific literature, including accession numbers to GenBankdatabase sequences, referred to herein establish the knowledge of thosewith skill in the art and are hereby incorporated by reference in theirentirety to the same extent as if each was specifically and individuallyindicated to be incorporated by reference. Any conflict between anyreference cited herein and the specific teachings of this specificationshall be resolved in favor of the later. Likewise, any conflict betweenan art-understood definition of a word or phrase and a definition of theword or phrase as specifically taught in this specification shall beresolved in favor of the latter.

[0067] In a first aspect the invention provides a method for inhibitingthe expression of a gene in a cell comprising contacting the cell withan effective synergistic amount of an antisense oligonucleotide whichinhibits expression of the gene, and an effective synergistic amount ofa protein effector of a product of the gene. In certain embodiments ofthe first aspect of the invention, the cell is contacted with aneffective synergistic amount of at least one antisense oligonucleotideand/or at least one protein effector for an effective period of time.

[0068] As used herein for all aspects of the invention, the term“protein effector” denotes an active moiety capable of inhibiting theindicated protein or product of a gene at the protein level. Forexample, a “DNA MeTase protein effector” or a “protein effector of theproduct of a DNA MeTase gene” inhibits DNA MeTase at the protein level.By “product of a gene” is meant a protein or polypeptide, regardless ofsecondary modifications (eg., glycosylation, lipidation, orphosphorylation), encoded by the gene. The term protein effectortherefore includes, without limitation, specific enzyme inhibitors whichare capable of inhibiting activity of the indicated protein or productof a gene. A protein effector is a molecule that inhibits the activityof the indicated protein to a greater extent than it inhibits theactivity of any unrelated protein. Preferably, a protein effectorinhibits the indicated protein at least 5-fold, more preferably at least10-fold, even more preferably at least 50-fold, and most preferably atleast 100-fold more than it inhibits any unrelated protein.

[0069] The terms “effective synergistic amount” and “effective period oftime” are used to denote known concentrations of the antisenseoligonudeotide and of the protein effector and for periods of timeeffective to achieve the result sought. The effective synergistic amountof the antisense oligonucleotide and/or the effective synergistic amountof the protein effector is/are less than the amount(s) empirically foundnecessary to inhibit the gene when either the antisense oligonucleotideor the protein effector are used individually. In preferred embodiments,the combined inhibitory effect of the antisense oligonucleotide and theprotein effector according to the invention are more than additive,i.e., the combined inhibitory effect is greater than the expected totalinhibitory effect calculated on the basis of the sum of the individualinhibitory effects.

[0070] The antisense oligonucleotides according to all aspects of theinvention are complementary to a region of double-stranded DNA or of RNA(or a region at the intron/exon boundary of DNA or RNA) that encodes theproduct of the gene. For purposes of the invention, the term“oligonucleotide” includes polymers of two or more deoxyribonucleosides,ribonucleosides, or 2′-O-substituted ribonucleoside residues, or anycombination thereof. Preferably, such oligonucleotides have from about 6to about 100 nucleoside residues, more preferably from about 8 to about50 nucleoside residues, and most preferably from about 12 to about 30nucleoside residues. The nucleoside residues may be coupled to eachother by any of the numerous known internucleoside linkages. Suchinternucleoside linkages include without limitation phosphorothioate,phosphorodithioate, alkylphosphonate, alkylphosphonothioate,phosphotriester, phosphoramidate, siloxane, carbonate,carboxymethylester, acetamidate, carbamate, thioether, bridgedphosphoramidate, bridged methylene phosphonate, bridged phosphorothioateand sulfone internucleotide linkages. In certain preferred embodiments,these internucleoside linkages may be phosphodiester, phosphotriester,phosphorothioate, or phosphoramidate linkages, or combinations thereof.The term oligonucleotide also encompasses such polymers havingchemically modified bases or sugars and/ or having additionalsubstituents, including without limitation lipophilic groups,intercalating agents, diamines and adamantane. For purposes of theinvention the term “2′-O-substituted” means substitution of the 2′position of the pentose moiety with an —O-lower alkyl group containing1-6 saturated or unsaturated carbon atoms, or with an —O-aryl or allylgroup having 2-6 carbon atoms, wherein such alkyl, aryl or allyl groupmay be unsubstituted or may be substituted, e.g., with halo, hydroxy,trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl,carbalkoxyl, or amino groups; or such 2′ substitution may be with ahydroxy group (to produce a ribonucleoside), an amino or a halo group,but not with a 2′-H group.

[0071] Particularly preferred antisense oligonucleotides utilized inthis aspect of the invention include chimeric oligonucleotides andhybrid oligonucleotides.

[0072] For purposes of the invention, a “chimeric oligonucleotide”refers to an oligonucleotide having more than one type ofinternucleoside linkage. One preferred example of such a chimericoligonucleotide is a chimeric oligonucleotide comprising aphosphorothioate, phosphodiester or phosphorodithioate region,preferably comprising from about 2 to about 12 nucleotides, and analkylphosphonate or alkylphosphonothioate region (see e.g., Pederson etal. U.S. Pat. Nos. 5,635,377 and 5,366,878). Preferably, such chimericoligonucleotides contain at least three consecutive internucleosidelinkages selected from phosphodiester and phosphorothioate linkages, orcombinations thereof.

[0073] For purposes of the invention, a “hybrid oligonucleotide” refersto an oligonucleotide having more than one type of nucleoside. Onepreferred example of such a hybrid oligonucleotide comprises aribonucleotide or 2′-O-substituted ribonucleotide region, preferablycomprising from about 2 to about 12 2′-O-substituted nucleotides, and adeoxyribonucleotide region. Preferably, such a hybrid oligonucleotidewill contain at least three consecutive deoxyribonucleosides and willalso contain ribonucleosides, 2′-O-substituted ribonucleosides, orcombinations thereof (see e.g., Metelev and Agrawal, U.S. Pat. No.5,652,355).

[0074] The exact nucleotide sequence and chemical structure of anantisense oligonucleotide utilized in the invention can be varied, solong as the oligonucleotide retains its ability to inhibit expression ofthe gene of interest. This is readily determined by testing whether theparticular antisense oligonucleotide is active by quantitating the mRNAencoding a product of the gene, or in a Western blotting analysis assayfor the product of the gene, or in an activity assay for anenzymatically active gene product, or in a soft agar growth assay, or ina reporter gene construct assay, or an in vivo tumor growth assay, allof which are described in detail in this specification or in Ramchandaniet al. (1997) Proc. Natl. Acad. Sci. USA 94: 684 689.

[0075] Antisense oligonucleotides utilized in the invention mayconveniently be synthesized on a suitable solid support using well knownchemical approaches, including H-phosphonate chemistry, phosphoramiditechemistry, or a combination of H-phosphonate chemistry andphosphoramidite chemistry (i.e., H-phosphonate chemistry for some cyclesand phosphoramidite chemistry for other cycles). Suitable solid supportsinclude any of the standard solid supports used for solid phaseoligonucleotide synthesis, such as controlled-pore glass (CPG) (see,e.g., Pon, R. T. (1993) Methods in Molec. Biol. 20: 465-496).

[0076] In certain embodiments of all aspects of the invention, any ofthe antisense oligonucleotides may be operably associated with one ormore protein effectors. A preferred operable linkage is a hydrolyzableassociation. Preferably, the hydrolyzable association is a covalentlinkage between the antisense oligonucleotide and the proteineffector(s). Preferably, such covalent bonding is hydrolyzable byesterases and/or amidases. Examples of such hydrolyzable bonding areshown in PCT publication WO96/07392, which is hereby incorporated byreference. Phosphate esters are particularly preferred.

[0077] In certain preferred embodiments, the covalent linkage may bedirectly between the antisense oligonudeotide and the protein effectorso as to integrate the protein effector into the backbone.Alternatively, the covalent linkage may be through an extendedstructure. Linkages of this type may be formed by covalently linking theantisense oligonudeotide to the protein effector through coupling ofboth the antisense oligonucleotide and the protein effector to a carriermolecule such as a carbohydrate, a peptide or a lipid or a glycolipid.Other preferred operable linkages include lipophilic association, suchas formation of a liposome containing oligonudeotide and the proteineffector covalently linked to a lipophilic molecule and thus associatedwith the liposome. Such lipophilic molecules include without limitationphosphotidylcholine, cholesterol and phosphatidylethanolamine, andsynthetic neoglycolipids, such as syalyllacNAc-HDPE. In certainpreferred embodiments, the operable association may not be a physicalassociation, but simply a simultaneous existence in the body, forexample, when the antisense oligonucleotide is associated with oneliposome and the protein effector is associated with another liposome.

[0078] The method and compositions according to the invention are usefulfor a variety of purposes. For example, they can be used as “probes” ofthe physiological function of a gene product by being used to inhibitthe activity and/or expression of the gene product in an experimentalcell culture or animal system and to evaluate the effect of inhibitingsuch activity and/or expression. This is accomplished by administeringto a cell or an animal an antisense oligonucleotide which inhibitsexpression of a gene and a protein effector of a product of the geneaccording to the invention and observing any phenotypic effects. Thismethod according to the invention is preferable to traditional “geneknockout” approaches because it is easier to use, and can be used toinhibit the gene and/or a product of the gene at selected stages ofdevelopment or differentiation. For example, where the gene encodes DNAMeTase, the method according to the invention can serve as a probe totest the role of DNA methylation in various stages of development.

[0079] Finally, the methods and compositions according to the inventionare useful in therapeutic approaches to human diseases including benignand malignant tumors involving the modulation and/or the suppression ofgene expression. The anti-tumor utility of antisense oligonucleotidesaccording to the invention is described in detail elsewhere in thisspecification.

[0080] All of the aspects of the invention disclosed herein areapplicable to the synergistic inhibition of any target gene and is notlimited to any particular gene or gene product. More specifically, theinvention relates to the inhibition of any target gene by the concurrentor sequential inhibition of the same target gene at both the geneticlevel (i.e., at either the DNA or the mRNA level) and at the proteinlevel. As exemplified herein for DNA MeTase, histone deacetylase, andthymidylate synthase, such methods and compositions are useful for avariety of purposes. The invention results in an improved inhibitoryeffect, thereby reducing the effective concentrations of either or boththe gene level and the protein levels inhibitors required to obtain agiven inhibitory effect as compared to those necessary when eitherinhibitor is used individually.

[0081] Thus, the methods and compositions according to all aspects ofthe invention are useful in therapeutic approaches to human diseasesinvolving the modulation and/or suppression of gene expression of aparticular target gene. Particularly preferred disease targets include,without limitation, various cancers. The methods and compositionsaccording to the invention may also be used to activate silenced genesto provide missing gene functions and thus improve a given condition.For example, the methods and compositions of the invention are useful todownregulate and/or suppress abnormal oncogene expression and activitythereby inhibiting tumorigenesis.

[0082] In certain embodiments, each of the antisense oligonucleotide andthe protein effector is admixed with a pharmaceutically acceptablecarrier prior to contact with the cell. In certain embodiments, theantisense oligonucleotide and the protein effector are mixed togetherprior to contact with the cell.

[0083] In certain preferred embodiments of the first aspect of theinvention, the cell is contacted separately with each of the antisenseoligonucleotide and the protein effector. For example, the cell may becontacted with the antisense oligonucleotide prior to being contactedwith the protein effector. The cell may be contacted with an effectivesynergistic amount of one or more antisense oligonucleotides of theinvention, followed by contact with an effective synergistic amount ofone or more protein effectors of the invention. This is particularlyuseful where the gene encodes a DNA MeTase and where the contacted cellis desired to undergo apoptosis or be arrested in the S phase of thecell cycle.

[0084] In another example, the cell may be contacted with the proteineffector prior to being contacted with the antisense oligonucleotide.This is particularly useful where the gene encodes a DNA MeTase wherethe contacted cell is desired to be arrested in the G₁ phase of the cellcycle.

[0085] Furthermore, where the gene encodes, for example, DNA MeTase, themethods and compositions according to the invention may also be used toactivate silenced genes to provide a missing gene function and thusameliorate disease symptoms. For example, the diseases beta thalassemiaand sickle cell anemia are caused by aberrant expression of the adultbeta globin gene or of a mutated gene. Most individuals suffering fromthese diseases have normal copies of the fetal gene for beta globin.However, the fetal gene is hypermethylated and is silent. Activation ofthe fetal globin gene could provide the needed globin function, thusameliorating the disease symptoms. In addition, the methods andcompositions according to the invention may be used as gene therapytools to maintain, activate, and/or modulate the expression of exogenoussequences otherwise liable to inhibition by methylation.

[0086] Accordingly, in a certain embodiment of the first aspect of theinvention, the gene encodes DNA MeTase. Particularly preferrednon-limiting examples of antisense oligonucleotides complementary toregions of RNA or double-stranded DNA encoding DNA MeTase utilized inthe method according to the invention are shown in Table 1. DNA MeTaseRNA (see e.g., Yen et al. (1992) Nucl. Acids Res. 9: 2287-2291; Yoder etal. (1996) J. Biol. Chem. 271: 31092-31097; Bester et al. (1988) J. Mol.Bio. 203(4): 971-983) or double stranded DNA regions include, withoutlimitation, intronic sequences, untranslated 5′ and 3′ regions,intron-exon boundaries as well as coding sequences from the DNA MeTasegene (see Ramchandani et al. (1998) Biol. Chem. 379(4-5): 535-540).

[0087] Particularly preferred oligonucleotides have nucleotide sequencesof from about 13 to about 35 nucleotides which include the nucleotidesequences shown in Table 1. Yet additional particularly preferredoligonucleotides have nucleotide sequences of from about 13 to about 19nucleotides of the -nucleotide sequences shown in Table 1. TABLE 1 SEQID NO. SEQUENCE TARGET (*) 1 5′-AAG CAT GAG CAC CGT TCT CC-3′ 513-532 25′-TTC ATG TCA GCC AAG GCC AC-3′ 5218-5199 5 5′-GCT GTC TCT TTC CAA ATCTT-3′ 323-342 6 5′-TTT CTG TTA AGC TGT CTC TT-3′ 333-352 7 5′-TTC TCCTTC ACA CAT TCC TT-3′ 352-371 8 5′-CGT GCA AGA GAT TCA ATT TC-3′ 369-3889 5′-AAG TCA CAT AAC TGA TTC TT-3′ 409-428 10 5′-CTC GGA TAA TTC TTC TTTAC-3′ 643-662 11 5′-CCA GGT AGC CCT CCT CGG AT-3′ 456-475 12 5′-AGG GATTTG ACT TTA GCC AG-3′ 232-251 13 5′ TCC AAG GAC AAA TCT TTA TT-3′496-515 14 5′-CAT GAG CAC CGT TCT CCA AG-3′ 510-529 15 5′-ACG TCC ATTCAC TTC CCG GT-3′ 536-555 16 5′-TCA CTT CTT GCT TGC TTC CC-3′ 565-584 175′-GCT TGG TTC CCG TTT TCT AG-3′ 556-575 18 5′-CTA GAC GTC CAT TCA CTTCC-3′ 540-559 19 5′-ACT CTA CGG GCT TCA CTT CT-3′ 577-576 20 5′-TCT GCCATT CCC ACT CTA CG-3′ 589-608 21 5′-CAT CTG CCA TTC CCA CTC TA-3′591-610 22 5′-GGC ATC TGC CAT TCC CAC TC-3′ 593-612 23 5′-ATC GGA CTTGCT CCT CCT GG-3′ 650-669 24 5′-GGT GAC GGG AGG GCA GAA CT-3′ 5087-510625 5′ TGC CAG AAA CAG GGG TGA CG-3′ 5100-5119 26 5′-GTG CAT GTT GGG GATTCC TG-3′ 5121-5140 27 5′-GTG AAC GGA CAG ATT GAC AT-3′ 5159-5178 28 5′AGG CCA CAA ACA CCA TGT AC-3′ 5186-5205 29 5′-CGA ACC TCA CAC AAC AGCTT-3′ 5217-5236 30 5′-GAT AAG CGA ACC TCA CAC AA-3′ 5223-5242 31 5′-CTGCAC AAT TTG ATC ACT AA-3′ 5253-5272 32 5′-CAG AAA CAG GGG TGA CGG GA-3′5097-5116 33 5′-GCA CAA AGT ACT GCA CAA TT-3′ 5263-5282 34 5′-TCC AGAATG CAC AAA GTA CT-3′ 5271-5290

[0088] As used herein, a DNA MeTase protein effector preferably inhibitsDNA MeTase at least 5-fold, more preferably at least 10-fold, even morepreferably at least 50-fold, and most preferably at least 100-fold morethan it inhibits any unrelated protein.

[0089] In preferred embodiments, the DNA MeTase protein effector is amoiety capable of forming a stable adduct with DNA methyltransferase,thus mimicking the transient covalent intermediate complex believed tobe formed during methylation (see, e.g., Wu and Sanfi (1987) J. Biol.Chem. 262: 4778-4786). Preferable examples of DNA MeTase proteineffectors include without limitation nucleoside analogs such as5-aza-2′-deoxycytidine (5-aza-dC), 5-fluoro-2′-deoxycytidine,5-aza-cytidine (5-aza-C), or 5,6-dihydro-5-azacytidine or apharmaceutically acceptable salt thereof. A method of synthesizing5,6-dihydro-5-azacytidine from 5′-aza-cytidine is described in U.S. Pat.No. 4,058,602 which is hereby incorporated by reference in its entirety.Additional DNA MeTase protein effectors include the inhibitors of DNAmethyltransferase enzyme, including hairpin oligonucleotides, describedin PCT publication no. WO 97/44346 (PCT application no. PCT/IB97/00879).

[0090] In particularly preferred embodiments of all aspects of theinvention, the antisense oligonucleotide is in operable association witha protein effector. The term “operable association” includes anyassociation between the antisense oligonucleotide and the proteineffector which allows an antisense oligonucleotide to inhibit DNA MeTasegene expression and allows protein effector(s) to inhibit DNA MeTaseenzyme activity.

[0091] In preferred embodiments of the first aspect of the invention,the invention provides a method for inhibiting DNA MeTase in a cellcomprising contacting the cell with an effective synergistic amount ofan oligonucleotide which inhibits DNA MeTase expression and with aneffective synergistic amount of a DNA MeTase protein effector.

[0092] In certain preferred embodiments of the first aspect of theinvention, the gene encodes a DNA MeTase and the cell comprises a genewhose expression has been inactivated by methylation. Thus, expressionof the gene is promoted and/reactivated in the contacted cell.Preferably, the invention provides compositions and methods for thereactivation of a tumor suppressor gene which has been inactivated bymethylation in a cell, such as a neoplastic cell or a cell predisposedto become a neoplastic. By “neoplastic cell,” as used herein for allaspects of the invention, is meant a cell that shows aberrant cellgrowth, such as increased cell growth. A neoplastic cell may be ahyperplastic cell, a cell that shows a lack of contact inhibition ofgrowth in vitro, a tumor cell that is incapable of metastasis in vivo,or a cancer cell that is capable of metastasis in vivo. Any growth ofneoplatic cells, whether metastatic or benign, is referred to herein asa tumor or a tumor growth.

[0093] In certain preferred embodiments, where the gene encodes DNAMeTase, the invention provides compositions and methods for thereactivation of a tumor suppressor gene which has been inactivated bymethylation. In particularly preferred embodiments, the inventionprovides a method for the reactivation of a p16 tumor suppressor genewhich had been inhibited by methylation.

[0094] In certain preferred embodiments of this aspect of the invention,where the gene encodes a product not limited to DNA MeTase, methods andcompositions are provided for the modulation of target oncogenes suchas, for example, the mutant forms of the RAS oncogene family which havebeen implicated in as many as 75% of human pancreatic cancers (see, eg.,Rodenhuis et al. (1992) Semin. Cancer Biol. 3(4): 241-247; Brentnall etal. (1995) Cancer Res. 55(19): 4264-4267).

[0095] In addition, the methods and compositions according to theinvention may also be used to inhibit any number of target genes and/orproducts of these genes. Consequently, the methods and compositions ofthe invention are useful in therapeutic approaches to various humanconditions such as inflammation or asthma as discussed herein.

[0096] Accordingly, in a certain embodiment of the first aspect of theinvention, the gene encodes a histone deacetylase (HDAC). There areseveral related forms of histone deacetylases in the histone deacetylasefamily. The family includes HDAC-1, HDAC-2, HDAC-3, HDAC4, HDAC-5, andHDAC-6. Histone deacetylase activity is thought to modulate theaccessibility of transcription factors to enhancer and promoterelements, and functional histone deacetylases have been implicated as arequirement in cell cycle progression in both normal and neoplasticcells. Thus, in certain embodiments of this aspect of the invention, theinvention provides an inhibitor of a histone deacetylase (e.g., HDAC-1,HDAC-2, HDAC-3, HDAC4, HDAC-5, and HDAC-6) using an oligonucleotidewhich inhibits a gene encoding at least one HDAC, and a HDAC proteineffector capable of inhibiting a the activity and/or expression of atleast one HDAC such as, for example, trichostatin A (TSA), depudecin,trapoxin, suberoylanilide hydroxamic acid (SAHA), FR901228 (FujisawaPharmaceuticals), MS27-275 (Mitsui Pharmaceuticals), CI-994 (ParkeDavis), oxamflatin (Shionogi and Co.), and sodium butyrate. An HDACprotein effector preferably inhibits HDAC-1, HDAC-2, HDAC-3, HDAC4,HDAC-5, and/or HDAC-6 at least 5-fold, more preferably at least 10-fold,most preferably at least 50-fold, and most preferably at least 100-foldmore than it inhibits any unrelated protein. The antisenseoligonucleotides according to this aspect of the invention arecomplementary to regions of RNA or double-stranded DNA that encodeHDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, and/or HDAC-6 (see e.g., GenBankAccession Number U50079 for HDAC-1, GenBank Accession Number U31814 forHDAC-2, and GenBank Accession Number U75697 for HDAC-3). Particularly,preferred oligonucleotides have nucleotide sequences of from about 13 toabout 35 nucleotides which include the nucleotide sequences shown inTables 2 and 3. Yet additional particularly preferred oligonucleotideshave nucleotide sequences of from about 15 to about 26 nucleotides ofthe nucleotide sequences shown in Tables 2 and 3. TABLE 2 SEQ ID TARGETNO. SEQUENCE (**) 35 5′-GAG ACA GCA GCA CCA GCG GG-3′ 17-36 36 5′-ATGACC GAG TGG GAG ACA GC-3′ 21-49 37 5′-GGA TGA CCG AGT GGG AGA CA-3′31-50 38 5′-CAG GAT GAC CGA GTG GGA GA-3′ 33-52 39 5′-TGT GTT CTC AGGATG ACC GA-3′ 41-60 40 5′-GAG TGA CAG AGA CGC TCA GG-3′ 62-81 41 5′-TTCTGG CTT CTC CTC CTT GG-3′ 1504- 1523 42 5′-CTT GAC CTC CTC CTT GAC CC-3′1531- 1550 43 5′-GGA AGC CAG AGC TGG AGA GG-3′ 1565- 1584 44 5′-GAA ACGTGA GGG ACT CAG CA-3′ 1585- 1604 45 5′-CCG TCG TAG TAG TAA CAG ACT TT-3′138-160 46 5′-TGT CCA TAA TAG TAA TTT CCA A-3′ 166-187 47 5′-CAG CAA ATTATG AGT CAT GCG GAT TC-3′ 211-236

[0097] TABLE 3 SEQ ID TARGET NO. SEQUENCE (***) 50 5′-CTC CTT GAC TGTACG CCA TG-3′  1-20 51 5′-TGC TGC TGC TGC TGC TGC CG-3′ 121-141 525′-CCT CCT GCT GCT GCT GCT GC-Y 132-152 53 5′-CCG TCG TAG TAG TAG CAGACT TT-3′ 138-160 54 5′-TGT CCA TAA TAA TAA TTT CCA A-3′ 166-187 555′-CAG CAA GTT ATG GGT CAT GCG GAT TC-3′ 211-236 56 5′-GGT TCC TTT GGTATC TGT TT-3′ 1605- 1625

[0098] In another embodiment, the targeted gene of the invention encodesa thymidylate synthase. The thymidylate synthase-encoding gene can betargeted using an oligonucleotide which inhibits thymidylate synthaseand a thymidylate synthase protein effector capable of inhibitingthymidylate synthase activity such as, for example, the nucleosideanalog 5-fluorouracil (5-FU), Tomudex™ (Raltitrexed or Zeneca ZD1694),Zeneca ZD9331, Thymitaq™ (AG337, Agouron), AG331 (Agouron), Ly231514(Lilly), and BW1843U89. A thymidylate synthase protein effectorpreferably inhibits thymidylate synthase at least 5-fold, morepreferably at least 10-fold, most preferably at least 50-fold, and mostpreferably at least 100-fold more than it inhibits any unrelatedprotein. The antisense oligonucleotides according to this aspect of theinvention are complementary to regions of RNA or double-stranded DNAthat encode thymidylate synthase (see e.g., GenBank Accession NumberX02308). Particularly, preferred oligonucleotides have nucleotidesequences of from about 15 to about 35 nucleotides which include thenucleotide sequences shown in Table 4. Yet additional particularlypreferred oligonucleotides have nucleotide sequences of from about 13 toabout 20 nucleotides of the nucleotide sequences shown in Table 4. TABLE4 TARGET SEQ ID NO. SEQUENCE (****) 57 5′-GGA GGC AGG CCA AGT GGT CC-3′11-30 58 5′-CGG AGG CAG GCC AAG TGG TC-3′ 12-31 59 5′-GAC GGA GGC AGGCCA AGT GG-3′ 42-61 60 5′-ACG GAG GCA GGC GAA GTG GC-3′ 69-88 61 5′-GGACGG AGG CAG GCG AAG TG-3′ 71-90 62 5′-AAG CAC CCT AAA CAG CCA TT-3′1035-1054 63 5′-TTG AAA GCA CCC TAA ACA GC-3′ 1039-1058 64 5′-ACA ATATCC TTC AAG CTC CT-3′ 1059-1078 65 5′-CCT AAA GAC TGA CAA TAT CC-3′1070-1089 66 5′-AAT TAA TAA CTG ATA GGT CA-3′ 1163-1182 67 5′-CCA GTGGCA ACA TCC TTA AA-3′ 1183-1202 68 5′-CAC AGT TAC ATT TGC CAG TG-3′1197-1216 69 5′-TTA TGG AAA GAA CTG GCA CA-3′ 1213-1232 70 5′-CCT CAGCAT TGT CAG ATA CC-3′ 1260-1279 71 5′-TTC ATA ACC TCA GCA TTG TC-3′1267-1286 72 5′-ACA TTT CAT TCT CCT CAC TT-3′ 1289-1308 73 5′-CAT ACATTT CAT TCT CCT CA-3′ 1292-1311 74 5′-CCA ACC TTC TTT ATA AGT AC-3′1351-1370 75 5′-AAT TCA CCA ACC TTC TTT AT-3′ 1357-1376 76 5′-TTG AGGGAA TAG CTT GTG AA-3′ 1419-1438 77 5′-TTA CTC AGC TCC CTC AGA TT-3′1438-1457 78 5′-AAC ACT TCT TTA TTA TAG CA-3′ 1513-1532

[0099] Yet another target is dihydrofolate reductase (DHFR) using anoligonucleotide which inhibits DHFR expression and a DHFR proteineffector capable of inhibiting DHFR activity such as, for example,methotrexate (MTX) (e.g., for the treatment of tumors and/or autoimmuneinflammatory disorders). A DHFR protein effector preferably inhibitsDHFR at least 5-fold, more preferably at least 10-fold, most preferablyat least 50-fold, and most preferably at least 100-fold more than itinhibits any unrelated protein.

[0100] Additional preferred targets (e.g., for the treatment ofinflammation) according to this aspect of the invention include:cycloxygenase-2 (COX-2) using an oligonucleotide which inhibits (COX-2)expression and a COX-2 protein effector which inhibits (COX-2) activitysuch as, for example, Celecoxib (SC58635); telomerase using anoligonucleotide which inhibits telomerase expression and a proteineffector which inhibits telomerase activity such as, for example,3-azido-3-deoxythymidine; Topoisomerase I using an oligonucleotide whichinhibits Topoisomerase I expression and a Topoisomerase I proteineffector which inhibits Topoisomerase I activity such as, for example,Topotecan (SmithKline) or Camptothecin; Topoisomerase II using anoligonucleotide which inhibits Topoisomerase II expression and aTopoisomerase II protein effector which inhibits Topoisomerase IIactivity such as, for example, Etoposide (Bristol Myers Squibb); DNAPolymerase α using an oligonucleotide which inhibits DNA Polymerase aexpression and a DNA Polymerase α protein effector which inhibits DNAPolymerase α activity such as, for example, Ara-C; aromatase using anoligonucleotide which inhibits aromatase expression and a proteineffector which inhibits aromatase activity such as, for example,Letrozole (Femara, Novartis) anastrozole (Arimidex, Zeneca) vorozole(Rizivor); 5-α-reductase using an oligonucleotide which inhibits5-α-reductase expression and a 5-α-reductase protein effector whichinhibits 5-α-reductase activity such as, for example, FK143 (Fujissawa),Ly300502 (Eli Lilly and Co.); Neutrophil elastase (e.g., for thetreatment of inflammation) using an oligonucleotide which inhibitsNeutrophil elastase expression and a Neutrophil elastase proteineffector which inhibits Neutrophil elastase activity such as, forexample, ONO-5046.Na; farnesyltransferase using an oligonucleotide whichinhibits farnesyltransferase expression and a farnesyltransferaseprotein effector which inhibits farnesyltransferase activity such as,for example, L744832 (Merck), or BMS186511 (Bristol Myers Squibb);Cyclin kinases (CDKs) using an oligonucleotide which inhibits CDKsexpression and a CDKs protein effector which inhibits CDKs activity suchas for example Flavopiridol; the epidermal growth factor receptor (EGFR)using an oligonucleotide which inhibits EGFR expression and a EGFRprotein effector which inhibits EGFR activity such as, for example,PD153035 (Parke-Davis); Her-2/Neu using an oligonucleotide whichinhibits Her-2/Neu expression and a Her-2/Neu protein effector whichinhibits Her-2/Neu activity such as, for example, Tyrphostin, or AG825(Agouron); Leukotriene receptor LTD4 (e.g., for the treatment of asthma)using an oligonucleotide which inhibits Leukotriene receptor LTD4expression and a Leukotriene receptor LTD4 protein effector whichinhibits Leukotriene receptor LTD4 activity such as, for example, MK571(Merck); P-glycoprotein using an oligonucleotide which inhibitsP-glycoprotein expression and a P-glycoprotein protein effector whichinhibits P-glycoprotein activity such as, for example, PSC 833, acyclosporin A analog.

[0101] In a second aspect, the invention provides a method for treatinga disease responsive to inhibition of a gene. The method according tothis aspect of the invention includes administering to a mammal,including a human, which has at least one cell affected by the disease,of an antisense oligonucleotide which inhibits expression of the gene,and a therapeutically effective synergistic amount of a protein effectorof a product of the gene. The antisense oligonucleotide and the proteineffectors are as described for the first aspect according to theinvention. “A disease responsive to inhibition of a gene” is one whichis associated with altered activity, levels, or functions of the geneand/or a product of the gene. The symptoms of such a disease arealleviated and/or eliminated by the modulation of the activity of thegene or of a product of the gene. “A cell affected by the disease” is acell which has altered activity, levels, or functions of the gene and/ora product of the gene.

[0102] For example, “a disease responsive to DNA MeTase inhibition” isone which is associated with altered methylation pattern(s) or alteredDNA MeTase activity, levels, or functions. The symptoms of such adisease are alleviated and/or eliminated by the modulation of DNA MeTaseactivity. “A cell affected by a disease responsive to DNA MeTaseinhibition” is a cell which has altered methylation pattern(s) oraltered DNA MeTase activity, levels, or functions.

[0103] In certain embodiments of the second aspect of the invention, themammal is administered a therapeutically effective synergistic amount ofat least one antisense oligonucleotide and/or at least one proteineffector for a therapeutically effective period of time.

[0104] The terms “therapeutically effective synergistic amount” and“therapeutically effective period of time” are used to denote treatmentsat dosages and for periods of time effective to achieve the therapeuticresult sought. The therapeutically effective synergistic amount of theantisense oligonucleotide and/or the therapeutically effectivesynergistic amount of the protein effector is/are less than theamount(s) empirically found necessary to inhibit the gene when eitherthe antisense oligonucleotide or the protein effector are usedindividually. In preferred embodiments, the combined inhibitory effectof the antisense oligonucleotide and the protein effector according tothe invention are more than additive, i.e.,the combined inhibitoryeffect is greater than the expected total effect calculated on the basisof the sum of the individual effects. One of skill in the art willappreciate that such synergistic effect resulting in a lower effectiveconcentration of either the antisense oligonucleotide, the proteineffector or both may vary considerably depending on the tissue, organ,or the particular mammal or patient to be treated according to theinvention. Furthermore, one of skill will appreciate that thetherapeutically effective synergistic amount of either the antisenseoligonucleotide or the protein inhibitor may be lowered or increased byfine tuning and altering the amount of the other component. Theinvention therefore provides a method to tailor theadministration/treatment to the particular exigencies specific to agiven mammal or patient. As illustrated in the following examples,therapeutically effective synergistic ranges may be easily determined,for example, empirically by starting at relatively low amounts and bystep-wise increments with concurrent evaluation of inhibition. Inparticularly preferred embodiments, the therapeutic composition of theinvention is administered systemically at a sufficient dosage to attaina blood level of antisense oligonucleotide from about 0.01 μM to about20 μM and of protein effector from about 0.01 μM to about 10 μM. Inparticularly preferred embodiments, the therapeutic composition isadministered at a sufficient dosage to attain a blood level of antisenseoligonucleotide from about 0.05 μM to about 15 μM and of proteineffector from about 0.05 μM to about 10 μM. In even more preferredembodiments, the blood level of antisense oligonucleotide is from about0.1 μM to about 10 μM and the blood level of protein effector is fromabout 0.1 μM to about 7 μM. For localized administration, much lowerconcentrations than this may be effective. Preferably, a total dosage ofantisense oligonucleotide will range from about 0.1 mg to about 30 mgoligonucleotide per kg body weight per day, while, a total dosage ofprotein effector will range from about 0.01 mg to about 5 mg proteineffector per kg body weight per day. In more preferred embodiments, atotal dosage of antisense oligonucleotide will range from about 1 mg toabout 20 mg oligonucleotide per kg body weight per day, while, a totaldosage of protein effector will range from about 0.1 mg to about 4 mgprotein effector per kg body weight per day. In most preferredembodiments, a total dosage of antisense oligonucleotide will range fromabout 2 mg to about 10 mg oligonucleotide per kg body weight per day,while, a total dosage of protein effector will range from about 0.1 mgto about 1 mg protein effector per kg body weight per day. Inparticularly preferred embodiments, the therapeutically effectivesynergistic amount of antisense oligonucleotide is 0.5 mgoligonucleotide per kg body weight per day, and the effectivesynergistic amount of protein effector is 0.1 mg per kg body weight perday.

[0105] In certain embodiments, each of the antisense oligonucleotide andthe protein effector is admixed with a pharmaceutically acceptablecarrier prior to administration to the mammal. In certain embodiments,the antisense oligonudeotide and the protein effector are mixed togetherprior to administration to the mammal.

[0106] Each of the antisense oligonucleotides and the protein effectorsaccording to all aspects of the invention may optionally be formulatedwith any of the well known pharmaceutically acceptable carriers ordiluents. Pharmaceutically acceptable carriers and their formulationsare well-known and generally described in, for example, Remington'sPharmaceutical Sciences (18th Edition, ed. A. Gennaro, Mack PublishingCo., Easton, Pa., 1990). Formulations of the invention may furthercontain one or more DNA MeTase inhibitors and/or one or more additionalDNA MeTase antisense oligonucleotide(s), and/or one or more proteineffector(s), or it may contain any other pharmacologically active agent.If an antisense oligonucleotide is administered with a protein effector,both may be admixed together with a pharmaceutically acceptable carrier.Where the antisense oligonucleotide is administered separately from theprotein effector, each may be mixed with a pharmaceutically acceptablecarrier. It will understood that where the antisense oligonucleotide andthe protein effector are administered separately, the samepharmaceutically acceptable carrier need not be the same for both.Rather, the pharmaceutically acceptable carrier is dependent on theroute of administration of the antisense oligonucleotide and of theprotein effector.

[0107] The compositions of the invention may be administered by anyappropriate means. For example, the compositions of the invention may beadministered to an mammal within a pharmaceutically-acceptable diluent,carrier, or excipient, in unit dosage form according to conventionalpharmaceutical practice. Administration may begin before the mammal issymptomatic for a disease responsive to inhibition of a gene. Forexample, where the disease is responsive to DNA MeTase inhibition, suchas cancer, administration may begin before the animal is symptomatic.

[0108] Any appropriate route of administration may be employed,including, without limitation, parenteral intravenous, intra-arterial,subcutaneous, sublingual, transdermal, topical, intrapulmonary,intramuscular, intraperitoneal, by inhalation, intranasal, aerosol,intrarectal, intravaginal, or by oral administration. Therapeutics maybe in the form of liquid solutions or suspensions; for oraladministration, formulations may be in the form of tablets or capsules;and for intranasal formulations, in the form of powders, nasal drops, oraerosols. The compositions may be administered locally to the areaaffected by a disease responsive to inhibition of a gene. For example,where the disease is responsive to DNA MeTase inhibition and is acancer, the composition may be administered directly into the tumormass). The compositions of the invention may be administeredsystemically.

[0109] In certain preferred embodiments of the second aspect of theinvention, the antisense oligonucleotide and the protein effector areadministered separately to the mammal. For example, the antisenseoligonucleotide may be administered to the mammal prior toadministration to the mammal of the protein effector. The mammal mayreceive one or more dosages of antisense oligonucleotide prior toreceiving one ore more dosages of protein effector. Where the geneencodes a DNA MeTase, this administration schedule is particularlyuseful where the cell affected by the disease is desired to undergoapoptosis or be arrested in the S phase of the cell cycle. Such anadministration schedule may be useful, for example, where the geneencodes DNA MeTase and where the disease is an aggressive cellproliferative disease such as metastatic cancer.

[0110] In another example, the protein effector may be administered tothe mammal prior to administration of the antisense oligonucleotide. Themammal may receive one or more dosages of protein effector prior toreceiving one or more dosages of antisense oligonucleotide. Where thegene encodes a DNA MeTase, this administration schedule is particularlyuseful where the cell affected by the disease is desired to be arrestedin the G₁ phase of the cell cycle. Such an administration schedule maybe useful, for example, where the gene encodes DNA MeTase and where thedisease is associated with cells whose growth arrest, rather than death,is desired. One non-limiting example of such a disease istransplantation graft rejection, where the host's immune cells (e.g.,lymphocytes and leukocytes) proliferate in response to the foreign cellsin the transplanted graft. Arrest of growth of the host's immune cells,rather than the death of these cells by apoptosis, is desirable.

[0111] In certain preferred embodiments of the second aspect of theinvention, the gene encodes a DNA MeTase and the cell affected by thedisease comprises a gene whose expression has been inactivated bymethylation. In certain embodiments, expression of the gene is promoted,restored, and/or reactivated in the cell in the mammal to which has beenadministered a therapeutically effective synergistic amount of anantisense oligonucleotide and a therapeutically effective synergisticamount of a protein effector. In preferred embodiments, the gene is thep16^(ink4) tumor suppressor gene.

[0112] In certain embodiments of this aspect of the invention, the geneencodes a DNA methyltransferase. In certain embodiments, the proteineffector is selected from the group consisting of 5-aza-cytidine,5-aza-2′-deoxycytidine, 5-fluoro-2′-deoxycytidine and5,6-dihydro-5-azacytidine.

[0113] In certain embodiments of this aspect, the gene encodes a histonedeacetylase. In certain embodiments, the protein effector is selectedform the group consisting of trichostatin A, depudecin, trapoxin,suberoylanilide hydroxamic acid, FR901228, MS-27-275, CI-994, and sodiumbutyrate.

[0114] In certain embodiments of this aspect, the gene encodes athymidylate synthase. In certain embodiments, the protein effector isselected form the group consisting of 5-fluorouracil (5-FU), Tomudex,Raltitrexed, Zeneca ZD1694, Zeneca ZD9331, Thymitaq, AG331, Ly231514,and BW1843U89.

[0115] In a third aspect, the invention provides a method for inhibitingtumor growth in a mammal. The method according to this aspect of theinvention includes administering to a mammal, including a human, whichhas at least one neoplastic cell present in its body a therapeuticallyeffective synergistic amount of an antisense oligonucleotide whichinhibits expression of a gene involved in tumorigenesis, and atherapeutically effective synergistic amount of a protein effector of aproduct of the gene. As used herein, “a gene involved in tumorigenesis,”is a gene whose aberrant expression is associated with tumorigenesis.Exemplary genes involved in tumorigenesis include the genes encoding DNAmethyltransferases, histone deaceylases (all forms), and thymidylatesynthase. By “tumorigenesis” is meant the genetic and phenotypic eventsinvolved in the progression of a normal cell to become a neoplasticcell. The antisense oligonucleotide and the protein effectors are asdescribed for the first aspect according to the invention.Administration and dosages are as described for the second aspectaccording to the invention.

[0116] In certain embodiments of this aspect of the invention, the geneencodes a DNA methyltransferase. In certain embodiments, the proteineffector is selected from the group consisting of 5-aza-cytidine,5-aza-2′-deoxycytidine, 5-fluoro-2′-deoxycytidine and5,6-dihydro-5-azacytidine.

[0117] In certain embodiments of this aspect, the gene encodes a histonedeacetylase. In certain embodiments, the protein effector is selectedform the group consisting of trichostatin A, depudecin, trapoxin,suberoylanilide hydroxamic acid, FR901228, MS-27-275, CI-994, and sodiumbutyrate.

[0118] In certain embodiments of this aspect, the gene encodes athymidylate synthase. In certain embodiments, the protein effector isselected form the group consisting of 5-fluorouracil, Tomudex,Raltitrexed, Zeneca ZD1694, Zeneca ZD9331, Thymitaq, AG331, Ly231514,and BW1843U89.

[0119] In embodiments of the third aspect of the invention, the mammalis administered a therapeutically effective synergistic amount of atleast one antisense oligonucleotide and/or at least one protein effectorfor a therapeutically effective period of time. The terms,“therapeutically effective synergistic amount” and “therapeuticallyeffective period of time,” are as described in the second aspect of theinvention. In certain embodiments of the third aspect, each of theantisense oligonucleotide and the protein effector is admixed with apharmaceutically acceptable carrier prior to administration to themammal. In certain embodiments, the antisense oligonucleotide and theprotein effector are mixed prior to administration to the mammal.

[0120] In certain preferred embodiments of the third aspect of theinvention, the the antisense oligonucleotide and the protein effectorare separately administered to the mammal. For example, the antisenseoligonucleotide may be administered to the mammal prior to theadministration of the protein effector. This is desirable where the geneencodes a DNA MeTase and where the neoplastic cell in the mammal isdesired to undergo apoptosis or be arrested in the S phase of the cellcycle. This administration schedule is particularly useful, for example,in the treatment of an aggressive, metastatic tumor such as melanoma.

[0121] Alternatively, the protein effector may be administered to themammal prior to the administration of the antisense oligonucleotide.This schedule is desirable where the gene encodes a DNA MeTase and wherethe neoplastic cells in the mammal is desired to be growth arrested inthe G₁ phase of the cell cycle. This administration schedule isparticularly useful, for example, in the treatment of slower growingtumors (e.g., prostate cancer) or in the treatment of infirmed patientswhere the presence of apoptotic cells is not desirable.

[0122] In certain preferred embodiments of the third aspect of theinvention, where the gene encodes a DNA MeTase, the neoplastic cellfurther comprises a gene whose expression has been inactivated bymethylation. In certain embodiments, expression of the gene is promoted,restored, and/or reactivated in the neoplastic cell in the mammal towhich has been administered a therapeutically effective synergisticamount of an antisense oligonucleotide and a therapeutically effectivesynergistic amount of a protein effector. In preferred embodiments, thegene is the p16^(ink4) tumor suppressor gene.

[0123] In a fourth aspect, the invention provides an inhibitor of a genecomprising an antisense oligonucleotide which inhibits expression thegene in operable association with a protein effector of a product of thegene. In certain embodiments of this aspect of the invention, theantisense oligonucleotide is in operable association with two or moreprotein effectors.

[0124] The antisense oligonucleotide and protein effector of this aspectof the invention as as described for the first aspect according to theinvention.

[0125] In certain embodiments of the fourth aspect of the invention, thegene encodes a DNA methyltransferase. In certain embodiments, theprotein effector is selected from the group consisting of5-aza-cytidine, 5-aza-2′-deoxycytidine, 5-fluoro-2′-deoxycytidine and5,6-dihydro-5-azacytidine. In certain embodiments, the gene encodes ahistone deacetylase. In certain embodiments, the protein effector isselected form the group consisting of trichostatin A, depudecin,trapoxin, suberoylanilide hydroxamic acid, FR901228, MS-27-275, CI-994,and sodium butyrate. In certain embodiments, the gene encodes athymidylate synthase. In certain embodiments, the protein effector isselected form the group consisting of 5-fluorouracil, Tomudex,Raltitrexed, Zeneca ZD1694, Zeneca ZD9331, Thymitaq, AG331, Ly231514,and BW1843U89.

[0126] In a fifth aspect, the invention provides a pharmaceuticalcomposition comprising an inhibitor of a gene comprising an antisenseoligonudeotide in operable association with a protein effector. Incertain embodiments, the composition further comprises apharmaceutically acceptable carrier.

[0127] In a sixth aspect, the invention provides a method forinvestigating the role of a gene and/or a product of that gene incellular growth, including the growth of tumor cells. In the methodaccording to this aspect, the cell type of interest (e.g., a neoplasticcell) is contacted with a synergistic amount of an antisenseoligonucleotide which inhibits expression of the gene and a synergisticamount of a protein effector of a product of the gene, as described forthe first aspect according to the invention, resulting in inhibition ofexpression of the gene in the cell. The combinations described hereinmay be administered at different points in the cell cycle (e.g., at theG₁ phase, S phase, or G₂/M phase), or in conjunction with promoters orinhibitors of cell growth to determine the role of the gene (e.g., thegene encoding DNA MeTase) in the growth of the cell type of interest. Incertain embodiments, the cell is a neoplastic cell.

[0128] The following examples are intended to further illustrate certainpreferred embodiments of the invention and are not limiting in nature.Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of this invention, and are coveredby the following claims.

EXAMPLE 1 Selection of Antisense Oligonucleotides Capable of InhibitingMeTase Expression in Neoplastic cells

[0129] To identify antisense oligodeoxynucleotides capable of inhibitingDNA MeTase gene expression in human neoplastic cells, 85phosphorothioate oligodeoxynucleotides (each 20 base pairs in length)bearing sequences complimentary to the 5′ and 3′ regions of human DNAMeTase mRNA and sequences complimentary to intronexon boundaries weresynthesized and screened for antisense activity. As shown in FIG. 1,the-two DNA MeTase mRNA regions were identified to be highly sensitiveto antisense inhibition were targeted by MG88 having the sequence 5′-AAGCAT GAG CAC CGT TCU CC-3′ (SEQ ID NO:1) (this oligonudeotide is targetedto the DNA MeTase mRNA 5′ UTR at nucleotides 532 to 513) and MG98 havingthe sequence 5′-UUC ATG TCA GCC AAG GCC AC-3′ (SEQ ID NO:2) (thisoligonucleotide is targeted to the DNA MeTase mRNA 3′ UTR at nucleotides5218 to 5199). These oligonucleotides were chemically modified asfollows: A equals 2′-deoxyriboadenosine; C equals 2′-deoxyribocytidine;G equals 2′-deoxyriboguanosine; T equals 2′-deoxyribothymidine; A equalsriboadenosine; U equals uridine; C equals ribocytidine; and G equalsriboguanosine. The underlined bases were 2′-methoxyribose substitutednucleotides. Non-underlined bases indicate deoxyribose nucleosides. Thebackbone of each oligonucleotide consisted of a phosphorothioate linkagebetween adjoining nucleotides. MG88 and MG98 have IC₅₀ values of 40 nMand 45 nM for inhibition of DNA MeTase mRNA, respectively.

[0130] MG88 was next tested for an ability to inhibit DNA MeTase mRNA intwo human neoplastic cells, A549 (human non-small cell lung carcinomacells) and T24 (human bladder cancer cells). Cells were transfected withlipofectin only (6.25 μg/ml), or lipofectin plus 20, 40, or 80 nM ofMG88 or control oligonucleotide MG208 having the sequence 5′-AAC GAT CAGGAC CCT TGU CC-3′ (SEQ ID NO:4). MG208 was modified as follows: A equals2′-deoxyriboadenosine; C equals 2′-deoxyribocytidine; G equals2′-deoxyriboguanosine; T equals 2′-deoxyribothymidine; A equalsriboadenosine; U equals uridine; C equals ribocytidine; and G equalsriboguanosine. The underlined bases were 2′-methoxyribose substitutednucleotides. Non-underlined bases indicate deoxyribose nucleosides. Thebackbone of MG208 consisted of a phosphorothioate linkage betweenadjoining nucleotides.

[0131] The MG88 and MG208 oligonucleotides were diluted to the desiredconcentration from a 0.1 mM stock solution in the transfection media.Cells were exposed to oligonucleotide plus lipofectin (or lipofectinonly) for four hours on day 0 and four hours on day 1 (after each fourhour treatment, cells were returned to complete media). On day 2 (i.e.,48 hours after the first treatment on day 0), cells were harvested,whole cell lysates were prepared, and DNA MeTase protein levels wereanalyzed by Western blotting analysis with a DNA MeTase-specific rabbitpolyclonal antibody (generated according to standard techniques of aglutathione S-transferase fusion protein containing the first 10 kDaamino terminus from DNA methyltransferase protein). As shown in FIG. 2,treatment with MG88, but not control MG208, produced dose-dependentreduction in DNA MeTase protein levels. Equal loading of all lanes wasconfirmed by blotting for α-actin protein levels with an actin-specificrabbit polyclonal antibody (commercially available from Santa CruzBiotech., Santa Cruz, Calif.).

EXAMPLE 2 Inhibition of the p16 Tumor Suppressor by targeting DNA MeTase

[0132] The cyclin-dependent kinase inhibitor (CDKI) p16^(ink4A)regulates the transition from G₁ to S-phases of the cell cycle (Serranoet al. (1993) Nature 366: 704-707). Inactivation of p16^(ink4A) is oneof the most frequently observed abnormalities in human cancer (Serranoet al., supra). Transcriptional inactivation and associatedhypermethylation of the p16^(ink4A) promote region have also beenobserved in virtually all types of cancer (Gonzales-Zulueta et al.(1995) Cancer Res. 55: 4531-4535; Merlo et al. (1995) Nat. Med. 7:686-692; Costello et al. (1996) Cancer Res. 56: 2405-2410; Lo et al.,Cancer Res. 56: 2721-2725). To investigate the effect of specificallyreducing cellular DNA MeTase levels on the expression and methylationstatus of a silenced p16^(ink4A) gene, T24 human bladder cancer cellsthat contain a hypermethylated and silenced p16^(ink4A) gene weretransfected with 40 nM or 75 nM MG88 or control oligonucleotide MG208with 6.25 μg/ml lipofectin for four hours each day for up to 10consecutive days. On days 3, 5, 8, and 10, p16^(ink4A) protein levelswere then analyzed by immunoprecipitation followed by Western blottinganalysis with a p16^(ink4A) protein-specific antibody (PharMingen). HeLacells were used as a positive (+) control for p16^(ink4A) proteinexpression.

[0133] As shown in FIG. 3A, induction of expression of p16^(ink4A)protein was detected after 5 days of treatment with either 40 nM or 75nM of MG88, but not after treatment with MG208. Because MG88 has anantiproliferative effect, p16^(ink4A) levels were normalized to cellnumber (FIG. 3B), Thus, induction of p16^(ink4A) protein by MG88 wasboth dose-dependent and time dependent. p16^(ink4A) was not detected incells treated with either 40 nM or 75 nM of the mismatch control MG208or lipofectin alone (FIG. 3A).

[0134] Moreover, reactivation of p16^(ink4A) protein expression by MG88also caused accumulation of hypophosphorylated pRb and inhibition ofcell proliferation. p16^(ink4A) regulation progression through the G₁phase of the cell cycle by inhibiting cyclin-dependent kinaseCDK4-mediated phosphorylation of pRB such that the hypophosphrylatedform of Rb is associated with G₁/G₀ growth arrest (Serrano et al.,supra). Cell lysates of T24 cells which were reactivated to expressp16^(ink4A) protein following transfection with MG88 were analyzed byWestern blotting analysis for phosphorylated pRb showed a decrease inthe amount of phosphorylated forms of pRb, thus increasing the relativeabundance of the hypophosphorylated form of pRb (FIG. 4). These resultsdemonstrate that high levels of DNA MeTase in T24 cells activelysuppress p16^(ink4A) gene expression and that restoration of p16^(ink4A)expression functionality regulations downstream molecular targets, suchas pRb.

[0135] Next, to determine whether de novo methylation and silencing ofthe re-expressed p16^(ink4A) gene occurred when DNA MeTase returned tonormal levels, T24 cells were transfected for 10 consecutive days witheither 40 or 75 nM or MG88 or MG208. Because transfection of the cellswith 75 nM MG88 had a significant antiproliferative and cell deathinducing effect, only cells treated with the lower dose (40 nM) of MG88were used for the remainder of the experiment. Cell lysates of the T24cells transfected for ten days were analyzed by Western blottinganalysis for expression of DNA MeTase protein,p16^(ink4A) protein, andactin protein (as a control for equal loading) 3, 5, and 7 days afterMG88 transfection. As shown in FIG. 5, DNA MeTase protein levelsincreased in the absence of MG88 treatment and returned to controllevels between days 5-7 post-treatment (middle panel). The level ofp16^(ink4A) protein decreased steadily over the post-treatment perioduntil it was barely detectable at day 7 post-treatment (FIG. 5, upperpanel). FIG. 6 shows the inverse relationship between DNA MeTase levelsand p16^(ink4A) protein levels during and after treatment with MG88.Interestingly, the loss of p16^(ink4A) protein expression began at day14 after DNA MeTase levels returned to near control levels. This lagsuggests that elevated levels of DNA MeTase over several rounds ofreplication are required to methylate and inactivate p16^(ink4A) geneexpression. That the inactivation and de novo methylation of p16^(ink4A)are coincident with elevated levels of the DNA MeTase (Dnmt1) suggeststhat it may contribute to de novo methylation activity itself.

[0136] To identify changes in the methylation status of the p16^(ink4A)promoter induced by MG88 treatment, methylation specific PCR (MSP) andbisulfite genomic sequencing was performed as previously described(Caldas et al. (1994) Nat. Gen. 8: 27-32; Frommer et al. (1991) Proc.Nat. Acad. Sci. USA 89: 1827-2831). MSP of the p16^(ink4A) promoter wasperformed on T24 cells treated with 40 μM or 75 μM of MG88 or MG208 for3, 5, 9, or 10 days. Briefly, the Oncor p16 detection system was used(commercially available from Oncor, Gaithersburg, MD). PCR was performedin a total volume of 25 μl under the following conditions: 100 ngbisulfate-treated DNA (Oncor),10 mM Tris-HCl, pH 8.3, 50 nM KCl, 1.5 mMMgCl₂, 250 μM dNTPs, 80 ng of each of the following primers(5′-GTAGGTGGGGAGGAGTlTAGTTT-3′ sense (SEQ ID NO: 79) and5′-TCTAATAACCAACCAACCCCTAA-3′ antisense (SEQ ID NO: 80)) and 1 unit ofAmpliTaq Gold (Perkin-Elmer). The denaturation cycle was 95° C. for 12min followed by 35 cycles at 95° C. for 45 seconds, 60° C. for 45seconds, and 72° C. for 60 seconds, and an elongation cycle of 72° C.for 10 minutes. the PCR product (5 μl) was analyzed on a 2% agarose gel.The unmethylated (U) and demethylated (D) primers (commerciallyavailable from Oncor) were used at the same conditions as the specificprimers. PCR products were subcloned into PCR2.1 (commercially availableform Invitrogen, Carlsbad, Calif.), and sequenced to determinedemethylation of CpG sites in the p16 proximal promoter.

[0137] As shown in FIG. 7, MSP analysis with PCR primers specific formethylated p16^(ink4A) (M) or unmethylated p16^(ink4A) (U) revealed thatdemethylation of the p16^(ink4A) promoter region occurred as early asday 3 of MG88 treatment. Treatment with MG208 or lipofectin alone had noeffect on methylation of the p16^(ink4A) gene (FIG. 7). FIG. 8 showsthat by employing bisulfite genomic sequencing on days 0, 3, and 5 oftreatment with MG88 or MG208, fifteen CpG sites within the p16^(ink4A)promote were found to be methylated in untreated T24 cells. Inhibitionof the DNA MeTase by MG88 led to demethylation of 5 of fifteen CpG sitesby day 3 and demethylation at all 15 CpG sites by day 5 of treatment,whereas treatment with the control MG208 had no effect on p16^(ink4A)methylation status (FIG. 8). Three days after cessation of MG88treatment the p16^(ink4A) promoter shows significant re-methylation at13 of 15 sites reflecting either de novo methylation of these sites or arapid expansion of a less affected population (FIG. 8, bottom panel).

[0138] To study the effect of specific inhibition of DNA MeTase on cellgrowth, cellular proliferation rates were monitored both during thetreatment and post treatment periods to determine the duration of theeffect. T24 cells were treated for 0-5 days with lipofectin only, 75 nMMG88 or 75 nM MG208. During and following treatment, cells were counted.As can be seen in FIG. 9B, during the course of treatment, MG88dramatically inhibited cell proliferation, whereas treatment of cellswith the control MG208 caused only minimal growth inhibition relative tolipofectin treated cells. Inhibition of cell proliferation persisted forapproximately one week post-treatment, consistent with the finding thatp16^(ink4A) expression was maintained until 7 days after the last doseof MG88 (see FIGS. 5 and 6). To determine whether the transientre-expression of p16^(ink4A) and growth-inhibitory effects induced byshort term treatment with MG88 were due to the expansion of a lessaffected (less demethylated) population of cells within the treatedpopulation, or to rapid inactivation of p16^(ink4A) after MG88withdrawal, single cell clones were isolated after treatment. SeveralMG88 clones were, in fact, p16^(ink4A) negative, confirming that theMG88 treatment produced a mixed population of p16^(ink4A) positive andnegative cells. Methylation analysis by bisulfite sequencing of thep16^(ink4A) promoter of one of the MG88 positive clones, clone 4-5,thirty days following treatment with a five day treatment with MG88revealed that the p16^(ink4A) promoter region was completely nonmethylated at all CpG sites evaluated (FIG. 9A), demonstrating that evenshort term (5 day) inhibition of the DNA MeTase by MG88 could induceprolonged re-expression of a silenced tumor suppressor gene. As shown inFIGS. 9B and 9C, clone MG88 C4-5 initially grew slowly following a fiveday treatment with MG88 as compared to lipofectin clone C-5 (treated for5 days with lipofectin) and MG208 C2-4 (treated for 5 days with MG208);however between days 40 and 45 days post treatment, the growth rate ofMG88 C4-5 cells increased dramatically (FIG. 9C). Determination ofp16^(ink4A) protein levels in MG88 C4-5 cells revealed a significantdecrease at Day 49 (FIG. 9C, inserted Western blotting analysis). Lossof p16^(ink4A) expression after prolonged culture in the absence of MG88treatment suggests that the DNA MeTase targeted (thought to encodemaintenance DNA MeTase activity) may have de novo methyltransferaseactivity and over time can methylate and inactivate previouslyunmethylated actively expressing genes.

EXAMPLE 3 Inhibition of MeTase Rapidly Induces p21^(WAF1)

[0139] Another member of the cyclin-dependent kinase inhibitor (CDKI)family, p21^(WAF1), inhibits a wide range of cyclin/CDK complexesinvolved in G₁ and S phase progression (Tam et al. (1994) Cancer Res.54: 5816-5820; Baghdassarian and French (1996) Hematol. Cell Ther. 88:313-323; Gotz et al. (1996) Oncogene 13: 391-398). To investigatewhether DNA MeTase and p21^(WAF1) protein levels are linked by aregulatory pathway, p21^(WAF1) protein levels were measured in T24 cellsin which DNA MeTase levels had been incrementally reduced by MG88treatment. To do this, DNA MeTase, p21^(WAF1), and α-actin proteinlevels were measured by Western blotting analysis in T24 cells that hadbeen treated for either 24 hours (i.e., one 4 hour transfection time) or48 hours (i.e., two 5 hour transfections 24 hours apart) with either 40nM or 75 nM of either MG88 or MG208. As shown in FIG. 10A, p21^(WAF1)increased directly with the reduction in DNA MeTase, while neitherlipofectin nor MG208 had an effect on either DNA MeTase or p21^(WAF1)levels.

[0140] T24 cells were also treated for 24 hours (i.e., four hourtransfection) with lipofectin only, or lipofectin plus 20, 40, or 80 nMof MG88 or MG208. After treatment, cell lysates were prepared andanalyzed by Western blotting analysis with a-actin-specific antibody orp21-specific antibody.. As shown in FIG. 10B, DNA MeTase inhibitioninduced p21^(WAF1) in a dose dependent fashion as early as 24 hoursafter MG88 treatment, consistent with a role for p21^(WAF1) in theantiproliferation effect observed with MG88 treatment (see, e.g., FIGS.3B and 9B).

[0141] Next, to determine if p21^(WAF1) was induced at thetranscriptional level, RNase protection assay were performed on cellstreated with 40 nM of either MG88 or MG208 for either 24 or 48 hours.Total cellular RNA was isolated from the cells and analysed for p21mRNA, p27 mRNA, and p18 mRNA using the human cell cycle-2 (hcc-2)multiprobe RNase protection kit commercially available from PharMingen,San Diego, Calif.). RNA loading was determined to be equal using thesignals from two housekeeping genes, L32 and GAPDH. As shown in FIG. 11,no increase in p21^(WAF1) mRNA was observed in response to treatmentwith MG88, suggesting that post-translational regulation of p21^(WAF1)protein is involved.

[0142] This example demonstrates that a functional antagonism betweenDNA MeTase and p21^(WAF1) on cellular proliferation exists. Thus, highlevels of DNA MeTase found in transformed cells may regulateproliferation by reducing cellular p21^(WAF1) levels. High levels of DNAMeTase in transformed cell may also compete directly for the downstreamtarget PCNA, since human DNA MeTase can compete with p21^(WAF1) for PCNAbinding (Chuang et al. (1997) Science 277: 1996-2000).

EXAMPLE 4 Synergistic Reactivation of the p16 Tumor Suppressor

[0143] The purpose of this example is to illustrate the ability of themethods and compositions of the invention to restore the expression ofgenes which are inactivated by methylation such as, for example, the p16tumor suppressor as illustrated herein. For this purpose, one day beforetransfection, T24 cells (ATCC No. HTB4) were plated onto 10 cm plates at4×10⁵ cells/dish. At the time of transfection, cells were washed withphosphate buffered saline (PBS) and 5 ml of optimem media (Gibco-BRL)containing 6.25 μl/ ml lipofectin transfection reagent (Gibco-BRL) wasadded. The oligonucleotides used were: MG88 having the sequence 5′-AAGCAT GAG CAC CGT TCU CC-3′ (SEQ ID NO:1) (this oligonucleotide istargeted to the DNA MeTase mRNA 5′ UTR at nucleotides 532 to 513) andMG98 having the sequence 5′-UUC ATG TCA GCC AAG GCC AC-3′ (SEQ ID NO:2)(this oligonucleotide is targeted to the DNA MeTase mRNA 3′ UTR atnucleotides 5218 to 5199). Negative controls used were: MG207 having thesequence 5′-UUA ATG TAA CCT AAG GUC AA-3′ (SEQ ID NO:3) and MG208 havingthe sequence 5′-AAC GAT CAG GAC CCT TGU CC-3′ (SEQ ID NO:4). Theseoligonudeotides were chemically modified as follows: A equals2′-deoxyriboadenosine; C equals 2′-deoxyribocytidine; G equals2′-deoxyriboguanosine; T equals 2′-deoxyribothymidine; A equalsriboadenosine; U equals uridine; C equals ribocytidine; and G equalsriboguanosine. The underlined bases were 2′-methoxyribose substitutednucleotides. Non-underlined bases indicate deoxyribose nucleosides. Thebackbone of each oligonucleotide consisted of a phosphorothioate linkagebetween adjoining nucleotides. The oligonucleotides were diluted to thedesired concentration from a 0.1 mM stock solution in the transfectionmedia. After a four-hour incubation at 37° C. in a 5% CO₂ incubator, theplates were washed with PBS and 10 ml of fresh cell culture media wasadded. Following an additional two-hour incubation at 37° C., freshlyprepared 5-aza-2′-deoxycytidine was added to the tissue culture medium.Cells were transfected for a total of three days and split every otherday to ensure optimal transfection conditions. At the indicated timepoints, cells were harvested by trypsinization and pelleted bycentrifugation at 1100 rpm and 4° C. for five minutes. The cell pelletwas resuspended in PBS and counted on a Coulter Particle Counter todetermine the total cell number. Following a second centrifugation, thePBS was aspirated from the cell pellet and the pellet was frozen at 70°C.

[0144] Cell pellets were resuspended in 200 μl of cell lysis buffer (25mM Tris pH 7.5, 5 mM EDTA, 0.5% sodium deoxycholate, and 1% triton×100)supplemented with protease inhibitors: aprotinin, leupeptin, pepstatin,TLCK, and PMSF. The lysed cells were incubated on ice for 10 minutes andcell particulate matter was removed by centrifugation at 10,000 rpm for10 minutes at 4° C. The protein concentration for each sample wasdetermined using the Bio Rad Protein Assay and 600 μg of protein wasused for each immunoprecipitation. Anti-p16 antibody (Santa Cruz) wasadded, 2.5 μg/ml, and each sample was incubated at 4° C. for one hour ona rotary shaker. Each sample was incubated for an additional 45 minutesat 4° C. after the addition of 20 μl of equilibrated Protein G andwashed three times with lysis buffer containing no protease inhibitors.The Protein G pellet was resuspended in 15 μl of 2× gel loading buffer,containing β-mercaptoethanol and incubated at room temperature for 10minutes. After boiling for five minutes, samples were separated by gelelectrophoresis on a 4-20% polyacrylamide gradient gel and blotted ontoPVDF membrane (Amersham Life Sciences, Cleveland, Ohio). Each membranewas incubated overnight 1×TBST wash buffer (10 mM Tris pH 8.0, 150 mMNaCl, 0.05% Tween 20) supplemented with 5% milk. The membranes wereincubated with mouse anti-human p16 antibody (Pharmingen Mississauga,Ontario), 1 μg/ml, for sixty minutes at room temperature. Followingthree washes in 1×TBST, and a one-hour incubation with the secondaryantibody, goat anti-mouse IgG, membranes were washed two times in 1×TBSTand two times in 1×TBS, and chemiluminescence was performed. FIG. 12shows the Western blot analysis of T24 cells treated with variousconcentrations of 5-aza-dC from 0.1 μM to 0.75 μM, showing reactivationof p16 in the range of 0.3 μM to 0.75 μM 5-aza-dC. FIG. 13 shows theWestern blot analysis of T24 cells treated by the method of theinvention using antisense oligonucleotide MG88 and 5-aza-dC. Acomparison of the bands presence and intensity in FIG. 13 shows thatreactivation of the p16 gene is successfully achieved using acombination of the oligonucleotide and the protein effector according tothe invention at concentrations at which neither the oligonucleotide northe protein effector used alone would be effective (e.g., at as low as40 nM oligonucleotide MG88 and 0.1 μM 5-aza-dC).

[0145]FIG. 14 shows the Western blot analysis of T24 cells treated bythe method of the invention using antisense oligonucleotide MG98 and5-aza-dC. Once again, a comparison of the bands presence and intensityin FIG. 14 shows that reactivation of the p16 gene is successfullyachieved using a combination of the oligonucleotide and the proteineffector according to the invention at concentrations at which neitherthe oligonucleotide nor the protein effector used alone would beeffective (e.g., at as low as 40 nM oligonucleotide MG98 and 0.1 μM5-aza-dC).

[0146] The Western blotting analysis in FIG. 14 was subjected todensitometric analysis and normalized to the level of p16 expression inHeLa cells. The results of the densitometric analysis is shown in Table5. TABLE 5 Treatment of T24 cells Percentage of HeLa Cell Control (HeLacell control) 100 Lipofectin only 6 Lipofectin plus 0.1 μM 5-aza-dC 1140 nM MG207 1 40 nM MG207 plus 0.1 μM 5-aza-dC 3 40 nM MG98 4 40 nM MG98plus 0.1 μM 5-aza-dC 79

[0147] As Table 5 shows, the combination of both MG98 plus 5-aza-dC hasa much greater effect on p16 reactivation in T24 cells as compared toeither MG98 or 5-aza -dC alone.

[0148]FIG. 15 shows the Western blot analysis T24 cells treated by themethod of the invention using antisense oligonucleotide MG88 and5-aza-dC using even lower concentrations of oligonucleotide MG88. Acomparison of the bands presence and intensity in FIG. 15 shows thatreactivation of the p16 gene is successfully achieved using acombination of the oligonucleotide and the protein effector according tothe invention at concentrations at which neither the oligonucleotide northe protein effector used alone would be effective (e.g., at as low as20 nM oligonucleotide MG88 and 0.2 μM 5-aza-dC).

EXAMPLE 5 Synergistic Inhibition of Neoplastic cell Growth in Vitro

[0149] To illustrate the ability of the methods and composition of theinvention to inhibit DNA MeTase and to inhibit neoplastic cell growth ina synergistic fashion, T24 bladder carcinoma cells (ATCC No. HTB4;American Type Culture Collection, Manassas, Va.) or A549 human lungcarcinoma cells (ATCC No. CCL-185) were treated according to theinvention and their growth pattern observed and compared to that ofuntreated control cells. For this purpose, one day before transfection,cells were plated onto 10 cm plates at 4×10⁵ cells/dish. At the time oftransfection, cells were washed with phosphate buffered saline (PBS) and5 ml of Opti-MEM media (Gibco-BRL, Mississauga, Ontario) containing 6.25μl/ml lipofectin transfection reagent (Gibco-BRL Mississauga, Ontario)was added. The oligonudeotides used were: MG98 having the sequence5′-UUC ATG TCA GCC AAG GCC AC-3′ (SEQ ID NO:2) (this oligonucleotide istargeted to the DNA MeTase mRNA 3′UTR at nucleotides 5218 to 5199) andnegative control MG207 having the sequence 5′-UUA ATG TAA CCT AAG GUCAA-3′ (SEQ ID NO:3). These oligonucleotides were chemically modified asfollows: A equals 2′-deoxyriboadenosine; C equals 2′-deoxyribocytidine;G equals 2′-deoxyriboguanosine; T equals 2′-deoxyribothymidine; A equalsriboadenosine; U equals uridine; C equals ribocytidine; and G equalsriboguanosine. The underlined bases were 2′-methoxyribose substitutednucleotides. Non-underlined bases indicate deoxyribose nucleosides. Thebackbone of each oligonucleotide consisted of a phosphorothioate linkagebetween adjoining nucleotides.

[0150] The oligonucleotides were diluted to the desired concentrationfrom a 0.1 mM stock solution in the transfection media. After afour-hour incubation at 37° C. in a 5% CO₂ incubator, the plates werewashed with PBS and 10 ml of fresh cell culture media was added.Following an additional two-hour incubation at 37° C., freshly prepared5-aza-2′-deoxycytidine was added to the tissue culture medium. Cellswere transfected for a total of three days and split every other day toensure optimal transfection conditions. At the indicated time points,cells were harvested by trypsinization and pelleted by centrifugation at1100 rpm and 4° C. for five minutes. The cell pellet was resuspended inPBS and counted on a Coulter Particle Counter to determine the totalcell number. Following a second centrifugation, the PBS was aspiratedfrom the cell pellet and the pellet was frozen at 70° C. Treated anduntreated cells were analyzed by counting the cells according tostandard methodologies. The results of representative experiments areshown in FIGS. 16, 17, and 18. FIG. 16 shows a growth curve of T24 cellsthat had been treated for 1-7 days with lipofectin only (diamond); 0.05μM 5-aza-dC (square); 40 nM MG98 (triangle); 0.05 μM 5-aza-dC plus 40 nMMG98 (cross); 40 nM MG207 (star); or 0.05 μM 5-aza-dC plus 40 nM MG207(circle). FIG. 17 shows the number of T24 cells remaining after thecells had been treated for seven days with lipofectin only, 0.1 μM5-aza-dC, 40 nM MG207, 40 nM MG98, 0.1 μM 5-aza-dC plus 40 nM MG207, or0.1 μM 5-aza-dC plus 40 nM MG98. FIG. 18 shows a growth curve of A549cells that had been treated for 1-7 days with lipofectin only (diamond);40 nM MG98 (square); 0.05 μM 5-aza-dC plus 40 nM MG98 (triangle); 40 nMMG207 (X); or 0.05 μM 5-aza-dC plus 40 nM MG207 (star). These resultsshow that the oligonucleotides and protein effectors according to themethods of the invention are capable of inhibiting MeTase enzymaticactivity and neoplastic cell growth in a synergistic fashion resultingin an increased inhibitory effect as compared to that observed usingeither only the oligonucleotides or only the protein effectors. Theresults therefore attest to the ability of the invention to inhibit DNAMeTase using effective synergistic amounts of the antisenseoligonucleotide and/or of the protein effector according to theinvention.

EXAMPLE 6 Synergistic Effect on Neoplastic cells in Vivo

[0151] The purpose of this example is to illustrate the ability of themethods and compositions of the invention to treat diseases responsiveto DNA MeTase inhibition in mammals. This example further providesevidence of the ability of the methods and compositions of the inventionto inhibit tumor growth in a mammal. Eight to ten week old female BALB/cnude mice (Taconic Labs, Great Barrington, N.Y.) were injectedsubcutaneously in the flank area with 2×10⁶ preconditioned Colo 205human colon cancer cells (ATCC No. CCL-222). Preconditioning of thesecells was done by a minimum of three consecutive tumor transplantationsin the same strain of nude mice. Subsequently, tumor fragments ofapproximately 30 mgs were excised and implanted subcutaneously in mice,in the left flank area under Forene anesthesia (Abbott Labs., Geneva,Switzerland). When the tumors reached a mean volume of 100 mm³, the micewere treated intravenously for 7 consecutive days by daily bolousinfusion into the tail vein, with oligonucleotide saline preparationscontaining 0.16 mg/kg of antisense oligonucleotide and/or 5-aza-dC(Sigma, St. Louis, Mo.) also in saline preparations according to thepresent invention. The optimal final concentration of the oligonudeotideis established by dose response experiments according to standardprotocols. Tumor volume was calculated according to standard methodsevery second day post infusion (see methods, eg., in Meyer et al. (1989)Int. J. Cancer 43: 851-856). Treatment with the oligonudeotides andprotein effectors according to the methods of the invention caused asignificant reduction in tumor weight and volume relative to controlstreated with saline as shown in FIGS. 19, 20A, and 20B. FIG. 19 showsthat animals receiving only saline (diamond) or 0.5 mg/kg MG98 (square)for seven days showed tumor volume increase while those animalsreceiving 0.1 mg/kg 5-aza-dC only (triangle) showed less tumor volumeincrease. The most dramatic reduction in tumor volume was seen inanimals receiving both 0.5 mg/kg MG98 and 0.1 mg/kg 5-aza-dC (FIG. 19,X) for seven days.

[0152]FIGS. 20A and 20B show the results of a similar experiment, wheretumor volume is recorded for 25 days (i.e., 19 additional days afterfinal treatment. FIG. 20A shows a tumor volume growth curve versus timewhile FIG. 20B shows tumor volume in these mice on day 25. As shown inFIG. 20B, mice which had received both 0.5 mg/kg MG98 and 0.1 mg/kg5-aza-dC had statistically smaller tumors (p<0.05) than mice treatedwith saline only or 5-aza-dC only. In addition, the activity of DNAMeTase enzyme when measured is expected to be significantly reducedrelative to saline treated controls. These results show that theoligonucleotides and protein effectors according to the methods of theinvention are capable of inhibiting tumor growth in a synergisticfashion.

EXAMPLE 7 Different Synergistic Effects on Cell Cycle ProgressionInhibition with Scheduling Differences

[0153] The purpose of this example is to illustrate that the synergisticeffect on inhibition of cell cycle progression by a antisenseoligonucleotide of the invention combined with a protein effector of theinvention can be accomplished by different scheduling routines. In thisexample, T24 cells were used.

[0154] In Schedule A, the cells were transfected for four hours on day 0with 75 mM MG88. The next day, they were transfected again for fourhours with 75 mM MG88. The following day, they were treated with 1 μM5-aza-dC. After twenty-four hours, the media was replaced with freshmedia containing 1 μM aza-dC. For controls, the cells received either notreatment, treatment with only aza-dC (where the cells were untouchedfor the first two days), or treatment with only MG88 (where the cellswere untouched for the third and fourth days).

[0155] In schedule B, the cells were treated with 1 μM 5-aza-dC. Aftertwenty-four hours, the media was replaced with fresh media containing 1μM aza-dC. Twenty-four hours later, the cells were transfected for fourhours with 75 mM MG88. The next day, they were transfected again forfour hours with 75 mM MG88. For controls, the cells received either notreatment, treatment with only aza-dC (where the cells were untouchedfor the third and fourth days), or treatment with only MG88 (where thecells were untouched for the first and second days).

[0156] On the fifth day, the cells were harvested, and fixed andpermeabilized in ice-cold 70% ethanol, and stained with propidium iodideaccording to standard techniques (see, e.g., Ausubel et al, supra;Sambrook et al., supra). The stained cells were next analyzed byfluorescence activated cell sorter analysis (FACS analysis). Accordingto FACS analysis of cells stained with propidium iodide, cells cantypically be divided into four groups depending on the amount of DNAthey possess that is stained with the propidium iodide. Typically, 4Ncells (i.e., G₂/M phase cells that are about to divide) are at thefarthest right of a histogram. The next set of cells with the second tohighest staining are cells in S phase. The third most bright set ofcells is the 2N cells in the G₁ stage of cell cycle. Finally, the leastbright cells (i.e., those cells at the far left of a FACS histogram),are those that are undergoing apoptotic cell death.

[0157] As shown on FIG. 21, cells receiving both MG88 and 5-aza-dC inSchedule A (upper panel, far right) showed a decrease in the percentageof cells in the S phase of the cell cycle (27% in section M2) andincreases in the percentage of apoptosing cells (16% in section M1) andcells in the S phase (22% in section M3). In contrast, cells receivingboth MG88 and 5-aza-dC in Schedule B (FIG. 21, lower panel, far right)showed a more pronounced G₁ block (55% in section M2 in FIG. 21), andfewer 4N cells in the G₂/M population (13% in section M4). Notably,however, either Schedule A or Schedule B resulted in more inhibition ofcell cycle progression when both MG88 and 5-aza-dC were received, ascompared to cells receiving only MG88 or only 5-aza-dC (compare farright sections with middle sections in upper for Schedule A and lowerpanel for Schedule B).

EXAMPLE 8 Synergistic Effect on Inhibition of Thymidylate Synthase witha Combination of TS Antisense Oligonucleotide and TS Protein Effector

[0158] The enzyme thymidylate synthase (TS) catalyzes a critical step inthe synthesis of DNA and is especially crucial to neoplastic cellsundergoing uncontrolled proliferation. The example illustrates theability of a TS antisense oligonucleotide and a TS protein effector toact synergistically in cells to inhibit TS protein expression andinhibit the role of TS in cell cycle progression. The representativenonlimiting oligonucleotides of the invention used in this example wereMG2605, which has the following sequence: 5′ UUC ATA ACC TCA GCA TUG UC3′ (SEQ ID NO:71; this oligonucleotide is targeted to the thymidylatesynthase mRNA sequence provided in GenBank Accession No. X02308 atnucleotides 1267-1286) and control oligonucleotide MG2606, which has thefollowing sequence: 5′ GUC UTA AGC TCA ACA TUC UA 3′ (SEQ ID NO: 81).These oligonucleotides were chemically modified as follows: A equals2′-deoxyriboadenosine; C equals 2′-deoxyribocytidine; G equals2′-deoxyriboguanosine; T equals 2′-deoxyribothymidine; A equalsriboadenosine; U equals uridine; C equals ribocytidine; and G equalsriboguanosine. The underlined bases were 2′-methoxyribose substitutednucleotides. Non-underlined bases indicate deoxyribose nucleosides. Thebackbone of each oligonucleotide consisted of a phosphorothioate linkagebetween adjoining nucleotides.

[0159] The representative nonlimiting TS protein effector used in thisexample is the nucleoside analog 5-fluorouracil (5-FU).

[0160] To look at the ability of MG2605 to inhibit TS proteinexpression, T24 cells growing in culture were treated with lipofectinonly (6.25 μg/ml) or lipofectin plus 10 nM, 25 nM, or 50 nM of MG2605 orMG2606 for 72 hours (i.e., transfection for four hours per day for threeconsecutive days). After treatment, the cells were lysed and analyzed byWestern blotting analysis for thymidylate synthase protein levels usinga thymidylase synthase specific antibody (commercially available fromChemicon Int., Temecula, Calif.).

[0161] As shown in FIG. 22, T24 cells transfected for 72 hours with 25or 50 nM MG2605, but not control MG2606 showed diminished levels ofthymidylate synthase protein as compared to cells treated withlipofectin only. Equal loading of all wells is demonstrated byequivalent amounts of actin (FIG. 22).

[0162] To look at the ability of MG2605 to inhibit the role of TS incell cycle progression, T24 cells growing in culture were treated withlipofectin only (6.25 μg/ml) or lipofectin plus 25 nM of MG2605 orMG2606, or lipofectin plus 25 nM of MG2605 or MG2606 plus 500 nM 5-FUfor 72 hours (i.e., transfection for four hours per day for threeconsecutive days, with each transfection followed by incubation with 500nM of 5-FU). After treatment, the cells were processed for cell cycleanalysis by FACS analysis as described in Example 7.

[0163]FIG. 23 shows that T24 cells treated with 500 nM 5-FU incombination with MG2605 showed a greater number of cells arrested in theS phase (section M3; bottom panel), as compared to cells treated withonly MG2605 (3rd panel from top) or only 5-FU (4th panel from top).

[0164] In a similar experiment, T24 cells were transfected for fourhours with 25 nM of TS antisense oligonucleotide MG2605 or controloligonucleotide MG2606 at 0 and 24 hours. After each four hour exposureto oligonucleotide, cells were returned to serum-containing media. Someof the cells were returned to serum-containing media to which was added5 μM of 5-FU. At 72 hours, the cells were harvested, counted, and fixedand permeabilized in ice-cold 70% ethanol. The cells were then stainedwith propidium iodide and cell cycle analysis was performed by flowcytometry analysis as described in Example 7.

[0165]FIG. 24A shows the percentage of cells in G₁ phase (section M1), Sphase (section M2), and G₂/M phase (section M3) following treatment withnothing, MG2606 only, MG2605 only, 5-FU only, MG2606 plus 5-FU, orMG2605 plus 5-FU. Treatment with both MG2605 plus 5-FU resulted in agreater number of cells arrested in the S phase of the cell cycle andfewer cells in the G₁ phase of the cell cycle as compared to treatmentwith either MG2605 alone or 5-FU alone (FIG. 24A). Moreover, treatmentwith the combination of MG2605 plus 5-FU resulted in a lower numbercells as compared to the number of cells present following treatmentwith 5-FU only or MG2605 only (FIG. 24B).

[0166] This example demonstrates that expression of thymidylatesynthase, and its role in cell cycle progression, is inhibited in T24cells by the combination of a TS antisense oligonucleotide plus a TSprotein effector to a greater degree than that seen in T24 receivingeither the TS antisense oligonucleotide or the TS protein effectoralone.

EXAMPLE 9 Synergistic Effect on Inhibition of HDAC Activity with HDACAntisense Oligonucleotide and HDAC Protein Effector

[0167] Functional histone deacetylases (HDACS) have been implicated as arequirement in cell cycle progression in both normal and neoplasticcells. The example illustrates the ability of an HDAC antisenseoligonucleotide and a HDAC protein effector to act synergistically incells to inhibit HDAC protein expression and induce the cyclin-dependentkinase inhibitor (CDKI) family, p21^(WAF1).

[0168] To do this, the following representative, nonlimiting antisenseoligonucleotides targeting both HDAC-1 and HDAC-2 were used:

5′-CAG CAA ATT ATG GGT CAT GCG GAU UG-3′ (SEQ ID NO: 82);

5′-CAG CAA GTT ATG GGT CAT GCG GAU UG-3′ (SEQ ID NO:89);

5′-CAG CAA ATT ATG AGT CAT GCG GAU UG-3′ (SEQ ID NO: 90);

[0169] and

5′-CAG CAA GTT ATG AGT CAT GCG GAU UG-3′ (SEQ ID NO: 83).

[0170] This HDAC-1,2 antisense oligonudeotide (MG2610) was really a1:1:1:1 mixture of each oligonucleotide (i.e., 25% of each). The controloligonucleotide (MG2637) was likewise a 1:1:1:1 mixture of the followingfour different oligonucleotides:

5′-AAG GAA GTC ATG GAT GAT GCG CAU UG-3′ (SEQ ID NO: 84)

[0171] and

5′-AAG GAA ATC ATG AAT GAT GCG CAU UG-3′ (SEQ ID NO: 85).

5′-AAG GAA GTC ATG AAT GAT GCG CAU UG-3′ (SEQ ID NO: 86)

[0172] and

5′-AAG GAA ATC ATG GAT GAT GCG CAU UG-3′ (SEQ ID NO: 87).

[0173] These oligonudeotides were chemically modified as follows: Aequals 2′-deoxyriboadenosine; C equals 2′-deoxyribocytidine; G equals2′-deoxyriboguanosine; T equals 2′-deoxyribothymidine; A equalsriboadenosine; U equals uridine; C equals ribocytidine; and G equalsriboguanosine. The underlined bases were 2′-methoxyribose substitutednucleotides. Non-underlined bases indicate deoxyribose nucleosides. Thebackbone of each oligonucleotide consisted of a phosphorothioate linkagebetween adjoining nucleotides.

[0174] T24 cells were treated for 24 hours (i.e., 4 hours followed byincubation in fresh media for 20 hours) with lipofectin only, lipofectinplus 50 nM of MG2610 (i.e., HDAC-1,2 antisense oligonucleotide) orMG2637 control oligonucleotide, or lipofectin plus HDAC antisenseoligonucleotide or control oligonucleotide plus 10 ng/ml or 25 ng/mlTSA. The treated cells were then lysed and cellular lysates analyzed byWestern blotting analysis for p21^(WAF1) protein level. As shown in FIG.25, cells treated with the combination of HDAC-1,2 antisenseoligonucleotide plus TSA showed a greater increase in p21^(WAF1) proteinlevel as compared to cells treated with either HDAC-1,2 antisenseoligonucleotide or TSA alone.

[0175] This example demonstrates that treatment of cells with acombination of HDAC-1,2 antisense oligonucleotide plus an HDAC proteineffector had a synergistic ability to enhance p21^(WAF1) protein levelsas compared to treatment of cells with either HDAC-1,2 antisenseoligonucleotide alone or HDAC protein effector alone.

EXAMPLE 10 Synergistic Anti-Neoplastic Effect of Histone DeacetylaseAntisense Oligonucleotide and HDAC Protein Effector on Tumor Cells inVivo

[0176] The purpose of this example is to illustrate the ability of thehistone deacetylase antisense oligonucleotide and the HDAC proteineffector of the invention to inhibit tumor growth in a mammal. Thisexample further provides evidence of the ability of the methods andcompositions of the invention to inhibit tumor growth in domesticatedmammal. Eight to ten week old female BALB/c nude mice (Taconic Labs,Great Barrington, N.Y.) are injected subcutaneously in the flank areawith 2×10⁶ preconditioned A549 human lung carcinoma cells.Preconditioning of these cells is done by a minimum of three consecutivetumor transplantations in the same strain of nude mice. Subsequently,tumor fragments of approximately 30 mgs are excised and implantedsubcutaneously in mice, in the left flank area under Forene anesthesia(Abbott Labs., Geneva, Switzerland). When the tumors reaches a meanvolume of 100 mm³, one group of mice is treated daily witholigonucleotide saline preparations containing from about 0.1 mg toabout 30 mg per kg body weight of histone deacetylase antisenseoligonucleotide. A second group of mice is treated daily withpharmaceutically acceptable preparations containing from about 0.01 mgto about 5 mg per kg body weight of HDAC protein effector. Yet anothergroup of mice receives both the antisense oligonucleotide and the HDACprotein effector.

[0177] Of this third group, group A receives the antisenseoligonucleotide and the HDAC protein effector together simultaneouslyintravenously via the tail vein. Group B receives two infusions of theantisense oligonucleotide via the tail vein on Days 1 and 2, followed onDays 3 and 4 by two intravenous infusions of the HDAC protein effector.Group C receives two infusion of the HDAC protein effector followed bytwo infusions of the antisense oligonucleotide on Days 3 and 4.

[0178] Control groups of mice are similarly established which receive notreatment (e.g., saline only), a mismatch antisense oligonucleotideonly, a control compound that does not inhibit histone deacetylaseactivity, and mismatch antisense oligonucleotide with control compound.

[0179] Tumor volume is measured with calipers. Treatment with theantisense oligonucleotide plus the HDAC protein effector according tothe invention causes a significant reduction in tumor weight and volumerelative to controls. Preferably, the antisense oligonucleotide and theHDAC protein effector inhibit the expression and activity of the samehistone deacetylase.

1 90 1 20 DNA Artificial Sequence primer 1 aagcatgagc accgttctcc 20 2 20DNA Artificial Sequence primer 2 ttcatgtcag ccaaggccac 20 3 20 DNAArtificial Sequence primer 3 uuaatgtaac ctaaggucaa 20 4 20 DNAArtificial Sequence primer 4 aacgatcagg acccttgucc 20 5 20 DNAArtificial Sequence primer 5 gctgtctctt tccaaatctt 20 6 20 DNAArtificial Sequence primer 6 tttctgttaa gctgtctctt 20 7 20 DNAArtificial Sequence primer 7 ttctccttca cacattcctt 20 8 20 DNAArtificial Sequence primer 8 cgtgcaagag attcaatttc 20 9 20 DNAArtificial Sequence primer 9 aagtcacata actgattctt 20 10 20 DNAArtificial Sequence primer 10 ctcggataat tcttctttac 20 11 20 DNAArtificial Sequence primer 11 ccaggtagcc ctcctcggat 20 12 20 DNAArtificial Sequence primer 12 agggatttga ctttagccag 20 13 20 DNAArtificial Sequence primer 13 tccaaggaca aatctttatt 20 14 20 DNAArtificial Sequence primer 14 catgagcacc gttctccaag 20 15 20 DNAArtificial Sequence primer 15 acgtccattc acttcccggt 20 16 20 DNAArtificial Sequence primer 16 tcacttcttg cttgcttccc 20 17 20 DNAArtificial Sequence primer 17 gcttggttcc cgttttctag 20 18 20 DNAArtificial Sequence primer 18 ctagacgtcc attcacttcc 20 19 20 DNAArtificial Sequence primer 19 actctacggg cttcacttct 20 20 20 DNAArtificial Sequence primer 20 tctgccattc ccactctacg 20 21 20 DNAArtificial Sequence primer 21 catctgccat tcccactcta 20 22 20 DNAArtificial Sequence primer 22 ggcatctgcc attcccactc 20 23 20 DNAArtificial Sequence primer 23 atcggacttg ctcctcctgg 20 24 20 DNAArtificial Sequence primer 24 ggtgacggga gggcagaact 20 25 20 DNAArtificial Sequence primer 25 tgccagaaac aggggtgacg 20 26 20 DNAArtificial Sequence primer 26 gtgcatgttg gggattcctg 20 27 20 DNAArtificial Sequence primer 27 gtgaacggac agattgacat 20 28 20 DNAArtificial Sequence primer 28 aggccacaaa caccatgtac 20 29 20 DNAArtificial Sequence primer 29 cgaacctcac acaacagctt 20 30 20 DNAArtificial Sequence primer 30 gataagcgaa cctcacacaa 20 31 20 DNAArtificial Sequence primer 31 ctgcacaatt tgatcactaa 20 32 20 DNAArtificial Sequence primer 32 cagaaacagg ggtgacggga 20 33 20 DNAArtificial Sequence primer 33 gcacaaagta ctgcacaatt 20 34 20 DNAArtificial Sequence primer 34 tccagaatgc acaaagtact 20 35 20 DNAArtificial Sequence primer 35 gagacagcag caccagcggg 20 36 20 DNAArtificial Sequence primer 36 atgaccgagt gggagacagc 20 37 20 DNAArtificial Sequence primer 37 ggatgaccga gtgggagaca 20 38 20 DNAArtificial Sequence primer 38 caggatgacc gagtgggaga 20 39 20 DNAArtificial Sequence primer 39 tgtgttctca ggatgaccga 20 40 20 DNAArtificial Sequence primer 40 gagtgacaga gacgctcagg 20 41 20 DNAArtificial Sequence primer 41 ttctggcttc tcctccttgg 20 42 20 DNAArtificial Sequence primer 42 cttgacctcc tccttgaccc 20 43 20 DNAArtificial Sequence primer 43 ggaagccaga gctggagagg 20 44 20 DNAArtificial Sequence primer 44 gaaacgtgag ggactcagca 20 45 23 DNAArtificial Sequence primer 45 ccgtcgtagt agtaacagac ttt 23 46 22 DNAArtificial Sequence primer 46 tgtccataat agtaatttcc aa 22 47 26 DNAArtificial Sequence primer 47 cagcaaatta tgagtcatgc ggattc 26 48 20 DNAArtificial Sequence primer 48 aagcatgagc accgttcucc 20 49 20 DNAArtificial Sequence primer 49 uucatgtcag ccaaggccac 20 50 20 DNAArtificial Sequence primer 50 ctccttgact gtacgccatg 20 51 20 DNAArtificial Sequence primer 51 tgctgctgct gctgctgccg 20 52 20 DNAArtificial Sequence primer 52 cctcctgctg ctgctgctgc 20 53 23 DNAArtificial Sequence primer 53 ccgtcgtagt agtagcagac ttt 23 54 22 DNAArtificial Sequence primer 54 tgtccataat aataatttcc aa 22 55 26 DNAArtificial Sequence primer 55 cagcaagtta tgggtcatgc ggattc 26 56 20 DNAArtificial Sequence primer 56 ggttcctttg gtatctgttt 20 57 20 DNAArtificial Sequence primer 57 ggaggcaggc caagtggtcc 20 58 20 DNAArtificial Sequence primer 58 cggaggcagg ccaagtggtc 20 59 20 DNAArtificial Sequence primer 59 gacggaggca ggccaagtgg 20 60 20 DNAArtificial Sequence primer 60 acggaggcag gcgaagtggc 20 61 20 DNAArtificial Sequence primer 61 ggacggaggc aggcgaagtg 20 62 20 DNAArtificial Sequence primer 62 aagcacccta aacagccatt 20 63 20 DNAArtificial Sequence primer 63 ttgaaagcac cctaaacagc 20 64 20 DNAArtificial Sequence primer 64 acaatatcct tcaagctcct 20 65 20 DNAArtificial Sequence primer 65 cctaaagact gacaatatcc 20 66 20 DNAArtificial Sequence primer 66 aattaataac tgataggtca 20 67 20 DNAArtificial Sequence primer 67 ccagtggcaa catccttaaa 20 68 20 DNAArtificial Sequence primer 68 cacagttaca tttgccagtg 20 69 20 DNAArtificial Sequence primer 69 ttatggaaag aactggcaca 20 70 20 DNAArtificial Sequence primer 70 cctcagcatt gtcagatacc 20 71 20 DNAArtificial Sequence primer 71 ttcataacct cagcattgtc 20 72 20 DNAArtificial Sequence primer 72 acatttcatt ctcctcactt 20 73 20 DNAArtificial Sequence primer 73 catacatttc attctcctca 20 74 20 DNAArtificial Sequence primer 74 ccaaccttct ttataagtac 20 75 20 DNAArtificial Sequence primer 75 aattcaccaa ccttctttat 20 76 20 DNAArtificial Sequence primer 76 ttgagggaat agcttgtgaa 20 77 20 DNAArtificial Sequence primer 77 ttactcagct ccctcagatt 20 78 20 DNAArtificial Sequence primer 78 aacacttctt tattatagca 20 79 23 DNAArtificial Sequence primer 79 gtaggtgggg aggagtttag ttt 23 80 23 DNAArtificial Sequence primer 80 tctaataacc aaccaacccc taa 23 81 20 DNAArtificial Sequence primer 81 gucutaagct caacatucua 20 82 26 DNAArtificial Sequence primer 82 cagcaaatta tgggtcatgc ggauug 26 83 26 DNAArtificial Sequence primer 83 cagcaagtta tgagtcatgc ggauug 26 84 26 DNAArtificial Sequence primer 84 aaggaagtca tggatgatgc gcauug 26 85 26 DNAArtificial Sequence primer 85 aaggaaatca tgaatgatgc gcauug 26 86 26 DNAArtificial Sequence primer 86 aaggaagtca tgaatgatgc gcauug 26 87 26 DNAArtificial Sequence primer 87 aaggaaatca tggatgatgc gcauug 26 88 20 DNAArtificial Sequence primer 88 uucataacct cagcatuguc 20 89 26 DNAArtificial Sequence primer 89 cagcaagtta tgggtcatgc ggauug 26 90 26 DNAArtificial Sequence primer 90 cagcaaatta tgagtcatgc ggauug 26

What is claimed is:
 1. A method for inhibiting the expression of a genein a cell comprising contacting the cell with an effective synergisticamount of an antisense oligonucleotide which inhibits expression of thegene, and an effective synergistic amount of a protein effector of aproduct of the gene.
 2. A method for treating a disease responsive toinhibition of a gene in a mammal comprising administering to a mammal,including a human, which has at least one cell affected by the diseasepresent in its body, a therapeutically effective synergistic amount ofan antisense oligonucleotide which inhibits expression of the gene, anda therapeutically effective synergistic amount of a protein effector ofa product of the gene.
 3. A method for inhibiting tumor growth in amammal comprising administering to a mammal, including a human, whichhas at least one neoplastic cell present in its body, a therapeuticallyeffective synergistic amount of an antisense oligonucleotide whichinhibits expression of a gene involved in tumorigenesis, and atherapeutically effective synergistic amount of a protein effector of aproduct of the gene.
 4. The method of claim 1, 2, or 3, wherein theantisense oligonucleotide is in operable association with a proteineffector.
 5. The method of claim 1, 2, or 3, wherein the gene encodes aDNA methyltransferase.
 6. The method of claim 5, wherein the proteineffector is selected from the group consisting of 5-aza-cytidine,5-aza-2′-deoxycytidine, 5-fluoro-2′-deoxycytidine and5,6-dihydro-5-azacytidine.
 7. The method of claim 1, 2, or 3, whereinthe gene encodes a histone deacetylase.
 8. The method of claim 7,wherein the protein effector is selected form the group consisting oftrichostatin A, depudecin, trapoxin, suberoylanilide hydroxamic acid,FR901228, MS-27-275, CI-994, and sodium butyrate.
 9. The method of claim1, 2, or 3, wherein the gene encodes a thymidylate synthase.
 10. Themethod of claim 9, wherein the protein effector is selected form thegroup consisting of 5-fluorouracil, Tomudex, Raltitrexed, Zeneca ZD1694,Zeneca ZD9331, Thymitaq, AG331, Ly231514, and BW1843U89.
 11. The methodof claim 1, 2, or 3, wherein the antisense oligonucleotide has at leastone internucleotide linkage selected from the group consisting ofphosphorothioate, phosphorodithioate, alkylphosphonate,alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane,carbonate, carboxymethylester, acetamidate, carbamate, thioether,bridged phosphoramidate, bridged methylene phosphonate, bridgedphosphorothioate and sulfone internucleotide linkages.
 12. The method ofclaim 1, 2, or 3, wherein the antisense oligonucleotide is a chimericoligonucleotide comprising a phosphorothioate, phosphodiester orphosphorodithioate region and an alkylphosphonate oralkylphosphonothioate region.
 13. The method of claim 1, 2, or 3,wherein the antisense oligonucleotide comprises a ribonucleotide or2′-O-substituted ribonucleotide region and a deoxyribonucleotide region.14. The method of claim 1, wherein said cell is contacted with aneffective synergistic amount of at least one antisense oligonucleotidefor an effective period of time.
 15. The method of claim 2 or 3, whereinthe mammal is administered a therapeutically effective synergisticamount of at least one antisense oligonucleotide for a therapeuticallyeffective period of time.
 16. The method of claim 1, wherein said cellis contacted with an effective synergistic amount of at least oneprotein effector for an effective period of time.
 17. The method ofclaim 2 or 3, wherein the mammal is administered a therapeuticallyeffective synergistic amount of at least one protein effector for atherapeutically effective period of time.
 18. The method of claim 1,wherein each of the antisense oligonucleotide and the protein effectoris admixed with a pharmaceutically acceptable carrier prior tocontacting the cell.
 19. The method of claim 2 or 3, wherein each of theantisense oligonucleotide and the protein effector is admixed with apharmaceutically acceptable carrier prior to administration to themammal.
 20. The method of claim 1, wherein the antisense oligonucleotideand the protein effector are mixed prior to contacting the cell.
 21. Themethod of claim 2 or 3, wherein the antisense oligonucleotide and theprotein effector are mixed prior to administration to the mammal. 22.The method of claim 1, wherein the cell is contacted separately witheach of the antisense oligonucleotide and the protein effector.
 23. Themethod of claim 22, wherein the cell is contacted with the antisenseoligonucleotide prior to being contacted with the protein effector. 24.The method of claim 23, wherein the gene encodes a DNA methyltransferaseand wherein the contacted cell is induced to undergo apoptosis or isarrested in the S phase of the cell cycle.
 25. The method of claim 22,wherein the cell is contacted with the protein effector prior to beingcontacted with the antisense oligonucleotide.
 26. The method of claim25, wherein the gene encodes a DNA methyltransferase and wherein thecontacted cell is arrested in the G₁ phase of the cell cycle.
 27. Themethod of claim 2 or 3, wherein the antisense oligonudeotide and theprotein effector are separately administered to the mammal.
 28. Themethod of claim 27, wherein the antisense oligonudeotide is administeredto the mammal prior to the administration of the protein effector. 29.The method of claim 28, wherein the gene encodes a DNA methyltransferaseand wherein the cell in the mammal to which the antisenseoligonucleotide is administered prior to the administration of theprotein effector is induced to undergo apoptosis or is arrested in the Sphase of the cell cycle.
 30. The method of claim 27, wherein the proteineffector is administered to the mammal prior to the administration ofthe antisense oligonucleotide.
 31. The method of claim 30, wherein thegene encodes a DNA methyltransferase and wherein the cell in the mammalto which the protein effector is administered prior to theadministration of the antisense oligonucleotide is arrested in the G₁phase of the cell cycle.
 32. The method of claim 1, wherein the geneencodes a DNA methyltransferase and wherein the cell comprises a genewhose expression has been inactivated by methylation.
 33. The method ofclaim 32, wherein expression of the gene whose expression has beeninactivated by methylation is reactivated in the contacted cell.
 34. Themethod of claim 32, wherein the gene whose expression has beeninactivated by methylation is the p16^(ink4) tumor suppressor gene. 35.The method of claim 2 or 3, wherein the gene encodes a DNAmethyltransferase and wherein the cell comprises a gene whose expressionhas been inactivated by methylation.
 36. The method of claim 35, whereinexpression of the gene whose expression has been inactivated bymethylation is reactivated in the mammal to which has been administeredthe therapeutically effective synergistic amount of an antisenseoligonucleotide and the therapeutically effective synergistic amount ofa protein effector.
 37. The method of claim 35, wherein the gene whoseexpression has been inactivated by methylation is the p16^(ink4) tumorsuppressor gene.
 38. An inhibitor of a gene comprising an antisenseoligonucleotide which inhibits expression the gene in operableassociation with a protein effector of a product of the gene.
 39. Theinhibitor of claim 38, wherein the antisense oligonucleotide is inoperable association with two or more protein effectors.
 40. Theinhibitor of claim 38, wherein the gene encodes a DNA methyltransferase.41. The inhibitor of claim 40, wherein the protein effector is selectedfrom the group consisting of 5-aza-cytidine, 5-aza-2′-deoxycytidine,5-fluoro-2′-deoxycytidine and 5,6-dihydro-5-azacytidine.
 42. Theinhibitor of claim 38, wherein the gene encodes a histone deacetylase.43. The method of claim 42, wherein the protein effector is selectedform the group consisting of trichostatin A, depudecin, trapoxin,suberoylanilide hydroxamic acid, FR901228, MS-27-275, CI-994, and sodiumbutyrate.
 44. The inhibitor of claim 38, wherein the gene encodes athymidylate synthase.
 45. The inhibitor of claim 44, wherein the proteineffector is selected form the group consisting of 5-fluorouracil,Tomudex, Raltitrexed, Zeneca ZD1694, Zeneca ZD9331, Thymitaq, AG331,Ly231514, and BW1843U89.
 46. The inhibitor of claim 38, wherein theantisense oligonucleotide has at least one internucleotide linkageselected from the group consisting of phosphorothioate,phosphorodithioate, alkylphosphonate, alkylphosphonothioate,phosphotriester, phosphoramidate, siloxane, carbonate,carboxymethylester, acetamidate, carbamate, thioether, bridgedphosphoramidate, bridged methylene phosphonate, bridged phosphorothioateand sulfone internucleotide linkages.
 47. The inhibitor of claim 38,wherein the antisense oligonucleotide is a chimeric oligonucleotidecomprising a phosphorothioate, phosphodiester or phosphorodithioateregion and an alkylphosphonate or alkylphosphonothioate region.
 48. Theinhibitor of claim 38, wherein the antisense oligonudeotide comprises aribonucleotide or 2′-O-substituted ribonucleotide region and adeoxyribonucleotide region.
 49. A pharmaceutical composition comprisingthe inhibitor of claim
 38. 50. The composition of claim 49 furthercomprising a pharmaceutically acceptable carrier.